WO2021236328A1 - Génération de banque d'adnc - Google Patents

Génération de banque d'adnc Download PDF

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Publication number
WO2021236328A1
WO2021236328A1 PCT/US2021/030895 US2021030895W WO2021236328A1 WO 2021236328 A1 WO2021236328 A1 WO 2021236328A1 US 2021030895 W US2021030895 W US 2021030895W WO 2021236328 A1 WO2021236328 A1 WO 2021236328A1
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well
cdna
well plate
wells
mrna
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PCT/US2021/030895
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English (en)
Inventor
Millicent GABRIEL
Daniel Paul RAKIEC II
David A. Ruddy
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Novartis Ag
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Publication of WO2021236328A1 publication Critical patent/WO2021236328A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the present disclosure generally relates to sequencing library construction, and more specifically to an improved full-length complementary DNA (“cDNA”) library generation protocol for producing next generation sequencing (“NGS”) compatible cDNA in a greatly reduced reaction volume within a multi-well plate format.
  • cDNA complementary DNA
  • NGS next generation sequencing
  • cDNA libraries find use in a number of molecular biology applications including drug discovery, transcriptomics, and cellular assays. Construction of these libraries requires accurate, high-throughput methods of nucleic acid amplification.
  • existing cDNA library generation protocols are limited by their inefficiency. Specifically, existing protocols typically require large volumes of costly reagents and an extensive amount of time (e.g., multiple days), and laborious sample preparation in order to produce a useable library. Accordingly, there is a need for a more time efficient and cost efficient protocol for cDNA library generation.
  • An example method includes, positioning a sample well plate within a liquid handling system, wherein the sample well plate includes a plurality of wells containing cells. After positioning the sample well plate within the liquid handling system, producing cell lysate in at least a first well of the plurality of wells using the cells contained in at least the first well, wherein the cell lysate includes messenger RNA (mRNA); purifying the mRNA in at least the first well of the plurality of wells; synthesizing cDNA in at least the first well using the purified mRNA; preparing the synthesized cDNA in at least the first well for amplification; and amplifying the prepared cDNA in at least the first well, wherein amplifying the prepared cDNA in at least the first well increases an amount of cDNA in at least the first well.
  • mRNA messenger RNA
  • An example liquid handling system includes a sample well plate, wherein the sample well plate includes a plurality of wells containing cells; one or more pipetting devices; and a controller.
  • the controller includes memory storing one or more programs, and the one or more programs include instructions, which when executed by one or more processors of the controller, cause the liquid-handling system to produce cell lysate in at least a first well of the plurality of wells using the cells contained in at least the first well, wherein the cell lysate includes messenger RNA (mRNA); purify the mRNA in at least the first well of the plurality of wells; synthesize cDNA in at least the first well using the purified mRNA; prepare the synthesized cDNA in at least the first well for amplification; and amplify the prepared cDNA in at least the first well, wherein amplifying the prepared cDNA in at least the first well increases an amount of cDNA in at least the first well.
  • mRNA messenger RNA
  • Another example liquid-handling system includes means for producing cell lysate in at least a first well of a sample well plate that includes a plurality of wells using cells contained in at least the first well, wherein the cell lysate includes messenger RNA (mRNA); means for purifying the mRNA in at least the first well of the plurality of wells; means for synthesizing cDNA in at least the first well using the purified mRNA; means for preparing the synthesized cDNA in at least the first well for amplification; and means for amplifying the prepared cDNA in at least the first well, wherein amplifying the prepared cDNA in at least the first well increases an amount of cDNA in at least the first well
  • mRNA messenger RNA
  • FIG. 1 A is a block diagram illustrating an exemplary liquid-handling system.
  • FIG. IB illustrates an exemplary automated pipetting system.
  • FIG. 2 illustrates an exemplary process for an improved cDNA library generation protocol.
  • FIG. 3. is a flow chart illustrating an exemplary process for RNA sequence amplification.
  • FIG. 1A illustrates an exemplary liquid-handling system 100.
  • liquid-handling system 100 includes automated pipetting system 102, controller 104, centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and cooling module 116.
  • Automated pipetting system 102 includes deck 118, XYZ translator mechanism 120, pipette device(s) 122, and robotic arm(s) 124.
  • Deck 118 is a working surface of automated pipetting system 102 that is configured to hold various tools and hardware that automated pipetting system 102 utilizes for the processes described herein, such as well plate(s) 126, intermediate plate(s) 128, tip rack(s) 130, tube(s) 132, tub(s) 134, trough(s) 136, and magnetic stand(s) 138.
  • Well plate(s) 126 each have 384 wells and are configured to hold cell samples, reagents, and/or other liquids or materials.
  • well plate(s) 126 examples include 384-well hard-shell PCR plates and 384-well MIDI plates. In some examples, well plate(s) 126 have more or less than 384 wells (e.g., 96 wells).
  • Intermediate plate(s) 128 are configured to hold reagents and/or other liquids or materials, and are mainly used for mixing two or more reagents and/or other liquids.
  • Tip rack(s) 130 are configured to hole a plurality of pipette tips of various sizes (e.g., 50 microliter, 300 microliter, or 1000 microliter tips) to be used by pipette device(s) 122.
  • Tube(s) 132 are configured to hold reagents.
  • Tub(s) 134 are configured to hold purification beads (e.g., solid-phase reversible immobilization (SPRI) paramagnetic beads) and/or other purification reagents (e.g., a PEG solution).
  • Trough(s) 136 are configured to hold large amounts of reagent (e.g., 50 to 150 milliliters of reagent per trough).
  • magnetic stand(s) 138 are configured to hold one or more well plates 126 and to use a magnetic force to attract magnetic purification beads contained within the wells of the one or more well plates 126 (and any particles attached thereto) in order to separate the magnetic purification beads from supernatant that is also contained within the wells.
  • XYZ translator mechanism 120 is configured to translate pipette device(s) 122 (which are mounted on XYZ translator mechanism 120) in the X and Y dimensions, and also to raise and lower pipette device(s) 122 in the Z dimension. In doing so, XYZ translator mechanism 120 allows pipette device(s) 122 to access various tools and hardware that are positioned on deck 118 (e.g., well plate(s) 126, intermediate plate(s) 128, tip rack(s) 130, tube(s) 132, tub(s) 134, trough(s) 136, and/or magnetic stand(s) 138) and any cell samples, reagents, and/or other liquids or materials contained therein.
  • deck 118 e.g., well plate(s) 126, intermediate plate(s) 128, tip rack(s) 130, tube(s) 132, tub(s) 134, trough(s) 136, and/or magnetic stand(s) 138
  • Pipette device(s) 122 are configured to pick up and release cell samples, reagents, and/or other liquids or materials. Pipette device(s) 122 are also configured to hold cell samples, reagents, and/or other liquids or materials therein while XYZ translator mechanism 120 moves pipette device(s) 122 from one location within automated pipetting system 102 to another. For example, pipette device(s) 122 may release a specific volume of reagent into one or more wells of a well plate 126 (e.g., reagent that pipette device(s) 122 picked up from a tube 132 positioned on deck 118 and transferred to the well plate 126). As shown, XYZ translator mechanism 120 is communicatively connected to controller 104 (e.g., via one or more wired connections and/or one or more wireless connections (e.g., WiFi or Bluetooth)).
  • controller 104 e.g., via one or more wired connections and/or one or more
  • Robotic arm(s) 124 are configured to move/position tools and/or hardware included in liquid-handling system 100 (e.g., well plate(s) 126, intermediate plate(s) 128, tip rack(s) 130, tube(s) 132, tub(s) 134, trough(s) 136, and/or magnetic stand(s) 138).
  • robotic arm(s) 124 can move a well plate 126 from one area of deck 118 to another.
  • robotic arm(s) 124 can position a well plate 126 on a magnetic stand 138. Note, while FIG.
  • FIG. 1 A illustrates robotic arm(s) 124 as being integrated with automated pipetting system 102, in some examples, one or more robotic arms 124 are separate/independent modules that are not integrated into automated pipetting system 102 and are positioned outside of automated pipetting system 102. In some of these examples, the one or more robotic arms 124 are configured to access centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and/or cooling modulel 16. As shown, robotic arm(s) 124 are communicatively connected to controller 104 (e.g., via one or more wired connections and/or one or more wireless connections (e.g., WiFi or Bluetooth)).
  • controller 104 e.g., via one or more wired connections and/or one or more wireless connections (e.g., WiFi or Bluetooth)).
  • FIG. IB illustrates an exemplary automated pipetting system.
  • the exemplary automated pipetting system shown in FIG. IB is a version of automated pipetting system 102.
  • the version of automated pipetting system 102 includes XYZ translator mechanism 120 and a single pipette device 122 mounted thereon.
  • more than one pipette device 122 e.g., 2, 3, or 4 pipette devices
  • the simplified version of automated pipetting system 102 further includes a well plate 126 positioned on deck 118.
  • FIG. IB only illustrates a single well plate 126 positioned on deck 118, in some examples, more than one well plate 126 (e.g., 2, 3, or 4 well plates) is positioned on deck 118.
  • deck 118 is in no way limited by the illustration of deck 118 in FIG. IB, as the size and/or shape of deck 118 may vary (e.g., based on the components, hardware, and/or tools included in automated pipetting system 102 and/or the processes performed by automated pipetting system 102).
  • Example automated pipetting systems suitable for performing the functions of automated pipetting system 102 described herein include the Microlab® STAR® (Hamilton Company®), epMotion® 5070 TMX (Eppendorf®), PerkinElmer® JANUS® G3, and Beckman Coulter® Biomek® i7.
  • controller 104 is configured to control the various automated aspects of liquid-handling system 100.
  • controller 104 controls the movement of XYZ translator mechanism 120 and the picking up and/or releasing of cell samples, reagents, and/or other liquids or materials by pipette device(s) 122 described above (e.g., controller 104 controls a suctioning/pressure apparatus that allows pipette device(s) 122 to pick up and/or release cell samples, reagents, and/or other liquids or materials).
  • Controller 104 is a separate general-purpose computer that is communicatively connected (e.g., via one or more wired connections and/or wireless connections (e.g., WiFi and/or Bluetooth)) to various components of automated pipetting system 102 (e.g., XYZ translator mechanism 120 and robotic arm(s) 124) as well as to centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and cooling module 116. In some examples, controller 104 is not communicatively connected to one or more of centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and cooling module 116.
  • wired connections and/or wireless connections e.g., WiFi and/or Bluetooth
  • controller 104 is integrated into automated pipetting system 102 (instead of being a separate, general-purpose computer).
  • Controller 104 includes processor(s) 140, memory 142, and input/output (“I/O”) interface 144.
  • I/O interface 144 facilitates input and output processing for controller 104.
  • I/O interface 144 can facilitate input and output processing for one or more input devices (e.g., a keyboard, mouse, touchscreen, etc.) and/or one or more output devices (e.g., a display) that are communicatively connected to controller 104 (e.g., via one or more wired connections and/or wireless connections) and that an operator of liquid-handling system 100 can use to observe and control the processes and functions of controller 104.
  • Memory 142 includes random access memory (RAM), including but not limited to volatile RAM (e.g., DRAM, SRAM) and non-volatile RAM (e.g., NAND).
  • RAM random access memory
  • memory 142 further includes computer-readable storage media.
  • the computer-readable storage media are tangible and non-transitory.
  • memory 142 can include high speed random access memory and can also include non-volatile memory, such as one or more magnetic disk storage devices, flash memory devices, or other non-volatile solid-state memory devices.
  • the computer-readable storage media of memory 142 store one or more programs for execution by processor(s) 140, the one or more programs including instructions for performing any of the methods and processes described herein (e.g., with reference to FIGS. 2-3).
  • Centrifuge 106 is configured to centrifuge one or more well plates 126. Centrifuge 106 is communicatively connected to controller 104 (such that controller 104 is able to control centrifuge 106 based on, for example, pre-programmed centrifuge routines). As mentioned above, in some examples, centrifuge 106 is not communicatively connected to controller 104. In these examples, centrifuge 106 is separately controlled by one or more operators of liquid-handling system 100.
  • Orbital shaker 108 is configured to shake one or more well plates 126 (and thus the contents of the wells of the one or more well plates 126) via continuous orbital motion.
  • Orbital shaker 108 is communicatively connected to controller 104 (such that controller 104 is able to control orbital shaker 108 based on, for example, pre-programmed shaking routines).
  • controller 104 is able to control orbital shaker 108 based on, for example, pre-programmed shaking routines.
  • orbital shaker 108 is not communicatively connected to controller 104.
  • orbital shaker 108 is separately controlled by one or more operators of liquid-handling system 100.
  • Example orbital shakers suitable for performing the functions of orbital shaker 108 described herein include the QInstruments® BioShake® 5000.
  • Vortex mixer 110 is configured to hold one or more well plates 126 and mix the contents of the wells of the one or more well plates 126 via rapid circular motion.
  • Vortex mixer 110 is communicatively connected to controller 104 (such that controller 104 is able to control vortex mixer 110 based on, for example, pre-programmed vortexing routines).
  • controller 104 is able to control vortex mixer 110 based on, for example, pre-programmed vortexing routines.
  • vortex mixer 110 is not communicatively connected to controller 104.
  • vortex mixer 110 is separately controlled by one or more operators of liquid-handling system 100.
  • Thermal cycler 112 is configured to heat and/or cool one or more well plates 126 in discrete, pre-programmed steps. For example, while a well plate 126 is positioned within thermal cycler 112, thermal cycler 112 can heat its interior to a first predetermined temperature (e.g., 60 degrees Celsius) for a first predetermined period of time (e.g., 5 minutes) and subsequently cool its interior to a second predetermined temperature (e.g., 4 degrees Celsius) for a second predetermined period of time (e.g., 10 minutes).
  • Thermal cycler 112 is communicatively connected to controller 104 (such that controller 104 is able to control thermal cycler 112 based on, for example, pre-programmed thermal cycle routines).
  • thermal cycler 112 is not communicatively connected to controller 104.
  • thermal cycler 112 is separately controlled by one or more operators of liquid-handling system 100 (e.g., the one or more operators directly input the pre-programmed thermal cycler routines into thermal cycler 112 instead of through controller 104).
  • Microheating system 114 includes three microheating modules that are each configured to heat one or more well plates 126 contained therein for a predetermined period of time.
  • each microheating module of microheating system 114 can heat up to a first predetermined temperature (e.g., 100 degrees Celsius) and then hold that first predetermined temperature for an extended period of time (e.g., 20 minutes) while a well plate 126 is contained therein.
  • a first predetermined temperature e.g., 100 degrees Celsius
  • an extended period of time e.g. 20 minutes
  • Microheating system 114 is communicatively connected to controller 104 (such that controller 104 is able to individually control a temperature of each microheating module included in microheating system 114). As mentioned above, in some examples, microheating system 114 is not communicatively connected to controller 104. In these examples, the microheating modules included in microheating system 114 are separately controlled by one or more operators of liquid handling system 100 (e.g., the one or more operators directly set the temperature of the microheating modules instead of controller 104).
  • Example microheating modules suitable for performing the functions described herein include the Hamilton® Heater Shaker and the SciGene® TruTemp® Heating System.
  • microheating system 114 includes less than three microheating modules (e.g., one or two microheating modules). In other examples, microheating system 114 includes more than three microheating modules (e.g., four or five microheating modules).
  • Cooling modulel 16 is configured to cool the contents of one or more wells of a well plate 126. As shown in FIG. 1 A, cooling module 116 is communicatively connected to controller 104 (such that controller 104 is able to control a temperature of cooling module 116). In some examples, cooling module 116 is a cooling block that is configured to hold and cool one or more well plates 126.
  • Example cooling blocks suitable for performing the functions of cooling module 116 described herein include the INHECO® CP AC® Ultraflat cooling blocks and the INHECO® CP AC® Microplate cooling blocks.
  • cooling module 116 is a physical container (e.g., a plate, a bowl, or the like) that contains ice and/or one or more other cooling agents (e.g., frozen or semi-frozen gels). As mentioned above, in some examples, cooling module 116 is not communicatively connected to controller 104. In these examples, cooling module 116 is separately controlled by one or more operators of liquid-handling system 100. For example, one or more operators separately control cooling module 116 (e.g., control the temperature of cooling module 116) when cooling module 116 is a physical container that contains ice and/or one or more other cooling agents.
  • FIG. 1A illustrates automated pipetting system 102, controller 104, centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and cooling module 116 as separate and individual modules of liquid-handling system 100, in some examples, one or more of controller 104, centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and cooling module 116 are integrated into automated pipetting system 102.
  • centrifuge 106, orbital shaker 108, vortex mixer 110, thermal cycler 112, microheating system 114, and/or cooling module 116 can be positioned on deck 118 of automated pipetting system 102 such that robotic arm(s) 124 (that are integrated into automated pipetting system 102) are capable of accessing each of these modules (e.g., to position one or more well plates 126 onto one or more of the modules).
  • FIG. 2 illustrates an exemplary process for an improved cDNA library generation protocol.
  • Process 200 is merely exemplary. Thus, some operations in process 200 are, optionally, combined, the orders of some operations are, optionally, changed, and some operations are, optionally, omitted. In some examples, process 200 is performed by a system similar or identical to liquid-handling system 100, described above with reference to FIG. 1 A.
  • liquid-handling system 100 produces cell lysate in the wells of a first well plate 126 (which contains 384 wells (e.g., a 384-well Eppendorf LowBind PCR plate, a 384-well cell culture plate, or the like)), according to the following process.
  • the first well plate 126 contains less than 384 wells (e.g., 96 wells).
  • more than one well plate 126 is used for step 202.
  • cell lysate is still produced in a total number of 384 wells during this step.
  • liquid-handling system instead of producing cell lysate in the wells of a 384-well plate, liquid-handling system produces cell lysate in the wells of four 96-well plates (e.g., for a total number of 384 wells).
  • the first well plate 126 is four separate 96-well plates.
  • one or more pipette devices 122 add 25 microliters of room temperature a Lysis Reagent mixture to each well of the first well plate 126 (with each well containing 100-100,000 cells (e.g., human and/or animal cells)).
  • the type of cells contained in the wells of the first well plate 126 vary between wells.
  • some wells of the first well plate 126 may contain cells of a first cell line (e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like) while other wells of the plate contain cells of a second cell line.
  • a first cell line e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like
  • second cell line e.g., there is no limit to the number of cell lines that can be contained in the wells of the first well plate 126 (e.g., there can be a different cell line in each well of the plate).
  • the Lysis Reagent mixture added to each well is taken from a source (e.g., a tube 132 or an intermediate plate 128 positioned on deck 118) originally consisting of 12.8 milliliters of Prototype Lysis Reagent and 0.29 milliliters of 1-Thioglycerol solution.
  • a source e.g., a tube 132 or an intermediate plate 128 positioned on deck 118
  • the volume of a reagent mixture source will vary based on the number of wells contained in the first well plate 126 being used.
  • the source mixture described above will include 23.5 milliliters of Prototype Lysis Reagent and 0.53 milliliters of 1-Thioglycerol solution.
  • the variance of reagent volumes in a source mixture based on the number of wells of the well plate 126 being used also applies to all other reagent mixture source volumes described below (e.g., the DNase Reagent mixture source volume, the Stop Reagent mixture source volume, the FSA and SSIII mixture source volume, and the like). Equation (1) below represents the volume of a reagent that should be added to a reagent mixture source based on a number of wells contained in the well plate 126 being used.
  • volume of Reagent for Source Mixture (Ratio of Reagent in Mixture) x (Volume of Reagent for One Well) x (Number of Wells) x (15% Overage) x (Dead Volume)
  • one or more pipette devices 122 gently pipette the contents of each well up and down 5-10 times (the more adherent the cells in each well are, the more pipetting required). Liquid-handling system 100 then allows each well to incubate at room temperature for at least 10 minutes.
  • one or more pipette devices 122 add 12.5 microliters of DNase Reagent mixture to each well of the first well plate 126.
  • one or more pipette devices 122 add the 12.5 microliters of DNase Reagent mixture to a second, unused well plate 126 (instead of the first well plate 126 (e.g., a Coming 384 well flat bottom microplate)).
  • the one or more pipette devices 122 subsequently transfer 25 microliters of the contents of each well of the first well plate 126 to wells of the second well plate 126 (each of which already contain the 12.5 microliters of DNase Reagent mixture), and the remaining sub-steps described as being performed using the first well plate 126 are instead performed using this second well plate 126.
  • the DNase Reagent mixture added to each well is taken from a source (e.g., a tube 132 or a trough 136) originally consisting of 0.08 milliliters of DNase 1 (which itself is a mixture of 1 vial of DNase 1 (Lyophilized) and nuclease-free water), 0.38 milliliters of RNasin Plus RNase Inhibitor, and 7.07 milliliters of Prototype DNAse Reagent.
  • a source e.g., a tube 132 or a trough 136
  • DNase 1 which itself is a mixture of 1 vial of DNase 1 (Lyophilized) and nuclease-free water
  • RNasin Plus RNase Inhibitor 0.38 milliliters of RNasin Plus RNase Inhibitor
  • 7.07 milliliters of Prototype DNAse Reagent 7.07 milliliters of Prototype DNAse Reagent.
  • one or more pipette devices 122 adds 12.5 microliters of Stop Reagent mixture to each well.
  • the Stop Reagent mixture is taken from a source (e.g., a tube 132 or a trough 136) originally consisting of 0.09 milliliters of 1-Thioglycerol Solution and 7.43 milliliters of Prototype Stop Reagent.
  • a source e.g., a tube 132 or a trough 136
  • one or more pipette devices 122 gently pipette the contents of each well up and down 5 times. Liquid-handling system 100 then allows the first well plate 126 to incubate at room temperature for at least 10 minutes.
  • liquid-handling system 100 incubates the first well plate at 65 degrees Celsius within a microheating module of microheating system 114 instead incubating the first well plate 126 at room temperature (e.g., for recalcitrant cells). Then, one or more pipette devices 122 transfer 25 microliters of the contents of each of the wells of the first well plate 126 to corresponding wells of a second, unused well plate 126 (e.g., a 384-well Eppendorf LowBind PCR plate).
  • a second, unused well plate 126 e.g., a 384-well Eppendorf LowBind PCR plate.
  • one or more (e.g., each) of the wells of the second well plate 126 will contain approximately 25 microliters of cell lysate (e.g., the internal contents of the 100- 10,000 cells that were originally contained in the wells).
  • the cell lysate contains about 1 nanogram or less of mRNA (although the amount of mRNA in the 25 microliters of cell lysate ranges between approximately 0.1 to 10 nanograms of mRNA).
  • step 202 takes about 90 minutes or less to complete.
  • cDNA library generation protocol with the generation of cell lysate can provide several benefits over existing cDNA library generation protocols.
  • existing cDNA library generation protocols typically begin with total RNA instead of the contents of lysed whole cells.
  • using cell lysate as the starting material for cDNA library generation can be much more time efficient because it bypasses the additional, time consuming steps of having to isolate total RNA from cells.
  • using cell lysate as the starting material for cDNA library generation can make this improved cDNA library generation protocol more available to the service industry (both in terms of cost and required expertise), as members of the service industry will no longer need to generate total RNA based on cell samples when generating a cDNA library.
  • cell lysate as described above did not increase an amount of undesirable non-coding pre-mRNA introns that is typically found in the starting material for existing cDNA library generation protocols (e.g., total RNA).
  • cell lysate as a starting material more time efficient, it also provides a starting material having equivalent quality as the starting materials for existing protocols.
  • the second well plate 126 includes one or more positive control wells and/or one or more negative control wells.
  • a positive control well may contain a mixture of 200 nanograms of Universal Human Reference (“UHR”) RNA and distilled water (e.g., for a 8 nanogram/microliter concentration of RNA) whereas a negative control may contain only distilled water.
  • UHR Universal Human Reference
  • a negative control may contain only distilled water.
  • liquid-handling system 100 purifies and fragments mRNA contained in the wells of the second well plate 126, according to the following process.
  • one or more pipette devices 122 add 12.5 microliters of previously-vortexed (using vortex mixer 110), room temperature RNA Purification Beads (“RPB”) to each well.
  • RPB room temperature RNA Purification Beads
  • one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rotations per minute (“rpm”) for 2 minutes.
  • one or more robotic arms 124 position the second well plate 126 on a microheating module of microheating system 114 (which has been preheated to 65 degrees Celsius). Liquid-handling system 100 then allows the second well plate 126 to incubate within the microheating module for 5 minutes. After incubating the second well plate 126 for 5 minutes, one or more robotic arms 124 position the second well plate 126 on cooling module 116. Liquid-handling system 100 then and allows the second well plate 126 to incubate on cooling module 116 for 1 minute.
  • liquid-handling system 100 allows the second well plate 126 to incubate at room temperature for 5 minutes (e.g., while positioned on deck 118). After 5 minutes have passed, one or more robotic arms 124 position the second well plate 126 on a magnetic stand 138. Liquid-handling system 100 then allows the second well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear). One or more pipette devices 122 then remove and discard the supernatant from each well.
  • one or more robotic arms 124 remove the second well plate 126 from the magnetic stand 138 (e.g., and position the second well plate 126 on deck 118).
  • One or more pipette devices 122 then add 30 microliters of room temperature Bead Washing Buffer (“BWB”) to each well.
  • BWB room temperature Bead Washing Buffer
  • one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rpm for 2 minutes.
  • one or more robotic arms 124 once again position the second well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the second well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then remove and discard the supernatant from each well.
  • one or more robotic arms 124 remove the second well plate 126 from the magnetic stand 138 (e.g., and position the second well plate 126 on deck 118).
  • One or more pipette devices 122 then add 12.5 microliters of room temperature Elution Buffer (“ELB”) to each well of the second well plate 126.
  • ELB room temperature Elution Buffer
  • one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rpm for 2 minutes.
  • one or more robotic arms 124 position the second well plate 126 within a microheating module of microheating system 114 (which has been preheated to 80 degrees Celsius). Liquid-handling system 100 then allows the second well plate 126 to incubate within the microheating module for 2 minutes. After incubating the second well plate 126 for 2 minutes, one or more robotic arms 124 position the second well plate 126 on cooling module 116. Liquid-handling system 100 then allows the second well plate 126 to incubate on cooling module 116 for 1 minute.
  • one or more robotic arms 124 remove the second well plate 126 from cooling module 116 and position the second well plate 126 back on deck 118. Then, one or more pipette devices 122 add 12.5 microliters of room temperature Bead Binding Buffer (“BBB”) to each well. After the one or more pipette devices 122 have added the BBB to the wells, one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rpm for 2 minutes.
  • BBB room temperature Bead Binding Buffer
  • Liquid handling system 100 then allows the second well plate 126 to incubate at room temperature for 5 minutes (e.g., after one or more robotic arms 124 position the second well plate 126 deck 118). After incubating the second well plate 126, one or more robotic arms 124 once again position the second well plate 126 on a magnetic stand 138. Liquid-handling system 100 then allows the second well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear). One or more pipette devices 122 then remove and discard the supernatant from each well.
  • one or more robotic arms 124 remove the second well plate 126 from the magnetic stand 138 (e.g., and position the second well plate 126 on deck 118) and one or more pipette devices 122 add 30 microliters of room temperature Bead Washing Buffer (“BWB”) to each well. After one or more pipette devices 122 have added the BWB to the wells, one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rpm for 2 minutes. Then, after shaking the second well plate 126, one or more robotic arms 124 position the second well plate 126 on a magnetic stand 138.
  • BWB room temperature Bead Washing Buffer
  • Liquid-handling system 100 then allows the second well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then remove and discard the supernatant from each well.
  • the one or more robotic arms 124 remove the second well plate 126 from the magnetic stand 138 (e.g., and position the second well plate 126 on deck 118) and one or more pipette devices 122 add 4.875 microliters of room temperature Fragment, Prime, Finish Mix (“FPF”) to each well.
  • FPF room temperature Fragment, Prime, Finish Mix
  • one or more robotic arms 124 position the second well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the second well plate 126 at 4000 rpm for 2 minutes.
  • one or more robotic arms 124 position the second well plate 126 within a microheating module of microheating system 114 (which has been preheated to 94 degrees Celsius). Liquid-handling system 100 then allows the second well plate 126 to incubate within the microheating module for 8 minutes. After one or more robotic arms 124 remove the second well plate 126 from the microheating module, one or more robotic arms 124 position the second well plate 126 on centrifuge 106. Centrifuge 106 then briefly centrifuges the second well plate 126 (e.g., for 1 or 2 minutes).
  • step 204 takes about 285 minutes or less to complete.
  • one or more operators of liquid handling system 100 prepare liquid-handling system 100 (e.g., prepare robotics, tools, hardware, and/or reagents included in liquid-handling system 100). In these examples, the preparation time takes about 60 minutes or less on average.
  • liquid-handling system 100 synthesizes single-stranded cDNA using the purified and fragmented mRNA contained in the wells of the second well plate 126, according to the following process.
  • one or more robotic arms position the second well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the second well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear).
  • one or more pipette devices 122 transfer 4.25 microliters of supernatant from each well of the second well plate 126 to corresponding wells of a third, unused well plate 126 (a 384-well PCR plate).
  • one or more robotic arms 124 position the third well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the second well plate 126 at 168 times gravity for 10 seconds.
  • one or more pipette devices 122 add 2 microliters of a First Strand Synthesis Act D Mix (“FSA”) and Superscript III Reverse Transcriptase (“SSIII”) mixture to each well of the third well plate 126.
  • FSA and SSIII mixture is taken from a source (e.g., a tube 132 or a trough 136) originally consisting of 205 microliters of previously-vortexed FSA (vortexed using vortex mixer 110) and 50 milliliters of SSIII that have been mixed together via gentle up and down pipetting.
  • one or more pipette devices 122 mix the previously-vortexed FSA and SSIII within an intermediate plate 128 during one or more of the previous sub-steps of step 206 and/or step 204 (e.g., while allowing the second well plate 126 to sit on the magnetic stand 138 for 5 minutes at the beginning of step 206).
  • Using an intermediate plate to mix reagents as described above can expedite steps of process 200 and thus make the entire cDNA library generation process more time efficient.
  • liquid-handling system 100 uses Superscript IV Reverse Transcriptase (“SSIV”) or ProtoScript II Reverse Transcriptase for this sub-step instead of SSIII (with equivalent volume amounts).
  • one or more robotic arms 124 position the third well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the third well plate 126 at 4000 rpm for 2 minutes. Then, after shaking the third well plate 126, one or more robotic arms 124 position the third well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the third well plate 126 at 168 times gravity for 10 seconds. Then, after centrifuging the third well plate 126 for 10 seconds, one or more robotic arms 124 position the third well plate 126 within a first microheating module of microheating system 114 (which has been preheated to 25 degrees Celsius).
  • one or more robotic arms 124 remove the third well plate 126 from the first microheating module and position the third well plate 126 within a second microheating module of microheating system 114 (which has been preheated to 42 degrees Celsius). After the third well plate 126 has incubated within the second microheating module for 15 minutes, one or more robotic arms 124 remove the third well plate 126 from the second microheating module and position the third well plate 126 within a third microheating module of microheating system 114 (which has been preheated to 70 degrees Celsius). After the third well plate 126 has incubated within the third microheating module for 15 minutes, one or more robotic arms 124 remove the third well plate 126 from the third microheating module (e.g., and position the third well plate 126 on deck 118).
  • one or more robotic arms 124 position the third well plate 126 on a single microheating module of microheating system 114.
  • the single microheating module increases its temperature from 25 degrees Celsius to 42 degrees Celsius and lastly to 70 degrees Celsius for each stage of incubation described above.
  • the third well plate 126 remains positioned within the single microheating system as the temperature of the microheating module is increased (e.g., by controller 104 or one or more operators).
  • step 206 takes about 82 minutes or less to complete.
  • liquid-handling system 100 synthesizes double-stranded cDNA using the single-stranded cDNA contained in the wells of the third well plate 126, according to the following process.
  • one or more pipette devices 122 add 6.25 microliters of a Resuspension Buffer (“RSB”) and Second Strand Marking Master Mix (“SMM”) mixture to each well of the third well plate 126.
  • RBS Resuspension Buffer
  • SMM Second Strand Marking Master Mix
  • the RSB and SMM mixture is taken from a source (e.g., a tube 132 or a trough 136) originally consisting of 80 microliters of room temperature RSB and 320 microliters of previously-centrifuged SMM (centrifuged at 168 times gravity for 10 seconds) that have been mixed together (via orbital shaking at 4000 rpm for 2 minutes).
  • a source e.g., a tube 132 or a trough 136
  • previously-centrifuged SMM centrifuged at 168 times gravity for 10 seconds
  • the RSB and SMM are mixed together using an intermediate plate 128 (which is then shaken by orbital shaker 108) during one or more of the previous steps of process 200 (e.g., during step 206).
  • one or more robotic arms 124 position the third well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the third well plate 126 at 168 times gravity for 10 seconds.
  • one or more robotic arms 124 position the third well plate 126 within thermal cycler 112 (which has had its lid preheated to 30 degrees Celsius). While positioned within thermal cycler 112, the third well plate 126 undergoes a pre-programmed thermal cycle (e.g., stored on memory 142) of 16 degrees Celsius for 1 hour.
  • a pre-programmed thermal cycle e.g., stored on memory 142
  • one or more robotic arms 124 position the third well plate 126 on deck 118.
  • Liquid-handling system 100 then allows the third well plate 126 to sit at room temperature until the contents of the wells are brought to room temperature.
  • one or more pipette devices 122 add 22.5 microliters of previously-vortexed (or, in some examples, previously mixed via gentle up and down pipetting) AMPure XP beads (vortexed or pipetted until homogeneous) to each well of the third well plate 126.
  • one or more pipette devices 122 gently pipette the contents of each well up and down 30 times.
  • liquid-handling system 100 allows the third well plate 126 to incubate at room temperature for 5 minutes (e.g., while positioned on deck 118).
  • One or more robotic arms 124 then position the third well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the third well plate 126 at 168 times gravity for 10 seconds.
  • one or more robotic arms 124 position the third well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the third well plate 126 to sit on the magnetic stand 138 for about 5-7 minutes (or until the liquid content of the wells is clear).
  • One or pipette devices 122 then remove and discard the supernatant from each well (e.g., about 33.75 microliters of supernatant from each well).
  • liquid-handling system 100 After removing the supernatant from each well, and while the third well plate 126 continues to sit on the magnetic stand 138, liquid-handling system 100 “washes” the contents of each well by adding 30 microliters of 80% ethyl alcohol (“EtOH”) to each well using one or more pipette devices 122, allowing the third well plate 126 to incubate on the magnetic stand 138 for 30 seconds after adding the 80% EtOH, and then removing and discarding supernatant from each well using one or more pipette devices 122 (e.g., about 30 microliters of supernatant from each well). Liquid-handling system 100 performs this wash twice.
  • EtOH 80% ethyl alcohol
  • one or more pipette devices 122 remove residual EtOH from each well. Once the residual EtOH is removed, liquid-handling system 100 allows the contents of the wells to air-dry for 5 minutes (while the third well plate 126 continues to sit on the magnetic stand).
  • one or more robotic arms 124 remove the third well plate 126 from the magnetic stand 138 (e.g., and position the third well plate 126 on deck 118) and one or more pipette devices 122 subsequently add 5.375 microliters of room temperature RSB to each well. Then, one or more robotic arms 124 position the third well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the third well plate 126 at 4000 rpm for 2 minutes. After, one or more robotic arms 124 remove the third well plate 126 from orbital shaker 108 and position the third well plate 126 on centrifuge 106.
  • centrifuge 106 centrifuges the third well plate 126 at 168 times gravity for 10 seconds.
  • liquid-handling system 100 allows the third well plate 126 to incubate at room temperature for 2 minutes (e.g., while positioned on centrifuge 106 or while positioned on deck 118).
  • one or more robotic arms 124 position the third well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the third well plate 126 to sit on the magnetic stand 138 for about 5 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then transfer 5.375 microliters of supernatant from each well of the third well plate 126 to corresponding wells of a fourth, unused well plate 126 (a 384-well PCR plate or a 384-well MIDI plate).
  • step 208 takes about 86 minutes or less to complete.
  • one or more operators of liquid-handling system 100 seal the fourth well plate 126 (using a Microseal adhesive seal) and store the fourth well plate 126 at -25 degrees Celsius to -15 degrees Celsius for up to 7 days before positioning the fourth well plate 126 back into liquid handling system 100 so that liquid-handling system 100 continues onto step 210.
  • liquid-handling system 100 adenylates the 3’ ends of the double- stranded cDNA contained in the wells of the fourth well plate 126, according to the following process.
  • one or more pipette devices 122 add 3.75 microliters of previously-centrifuged (centrifuged at 168 times gravity for 10 seconds), room temperature A-Tailing Mix (“ATL”) to each well of the fourth well plate 126.
  • ATL room temperature A-Tailing Mix
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes.
  • one or more robotic arms 124 position the fourth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds.
  • one or more robotic arms 124 position the fourth well plate 126 within a first microheating module of microheating system 114 (which has been preheated to 37 degrees Celsius). After the fourth well plate 126 has incubated within the first microheating module for 30 minutes, one or more robotic arms 124 remove the fourth well plate 126 from the first microheating module and position the fourth well plate 126 within a second microheating module of microheating system 114 (which has been preheated to 70 degrees Celsius). After the fourth well plate 126 has incubated within the second microheating module for 5 minutes, one or more robotic arms 124 remove the fourth well plate 126 from the second microheating module and position the fourth well plate 126 within cooling module 116 (which has been precooled to 4 degrees Celsius). The fourth well plate 126 incubates within cooling module 116 for 1 minute. Then, one or more robotic arms 124 remove the fourth well plate 126 from cooling module 116 (e.g., and position the fourth well plate 126 on deck 118).
  • one or more robotic arms 124 position the second well plate 126 on a single microheating module of microheating system 114.
  • the single microheating module increases its temperature from 37 degrees Celsius to 70 degrees Celsius for the two stages of incubation described above.
  • the fourth well plate 126 remains positioned within the single microheating system as the temperature of the microheating module is increased (e.g., by controller 104 or one or more operators).
  • step 210 takes about 80 minutes or less to complete.
  • one or more operators of liquid-handling system 100 prepare liquid-handling system 100 (e.g., prepare robotics, hardware, tools, and/or reagents included in liquid-handling system 100). In these examples, the preparation time takes about 30 minutes or less on average.
  • liquid-handling system 100 ligates indexing adapters to the ends of the adenylated double-stranded cDNA contained in the wells of the fourth well plate 126, according to the following process.
  • one or more pipette devices 122 adds 0.625 microliters of room temperature RSB to each well of the fourth well plate 126.
  • one or more pipette devices 122 adds 0.625 microliters of room temperature Ligation Mix (“LIG”) to each well of the fourth well plate 126.
  • LIG room temperature Ligation Mix
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes. After shaking the fourth well plate 126, one or more robotic arms 124 position the fourth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the plate at 168 times gravity for 10 seconds. One or more robotic arms 124 then position the fourth well plate 126 within a microheating module of microheating system 114 (which has been preheated to 30 degrees Celsius). Liquid-handling system 100 then allows the fourth well plate 126 to incubate within the microheating module for 10 minutes.
  • one or more robotic arms 124 position the fourth well plate 126 on cooling module 116 (or in some examples, a similarly-cold surface). Liquid-handling system 100 then allows the fourth well plate 126 to incubate on cooling module 116 for lto 2 minutes.
  • one or more pipette devices 122 add 1.25 microliters of previously-centrifuged (centrifuged at 168 times gravity for 10 seconds) Stop Ligation Buffer (“STL”) to each well of the fourth well plate 126. Cooling the contents of the wells and adding STL to the wells both slow/stop the ligation process.
  • Existing cDNA library generation protocols typically add STL to the wells of a well plate prior to cooling the contents of a well plate However, adding STL to all of the wells of a well plate can take several minutes (e.g., 30 minutes).
  • cooling the contents of the wells while adding the STL is advantageous because the contents of many of the wells would continue undergoing the ligation process while waiting for STL if the contents of those wells were not also being cooled at the same time.
  • cooling the contents of the wells while adding STL to the wells ensures that the contents of each well undergo the ligation process for a similar (e.g., the same) amount of time. .
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes. Then, one or more robotic arms 124 position the fourth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds. Next, one or more pipette devices 122 add 10.5 microliters of previously-vortexed (or, in some examples, previously mixed via gentle up and down pipetting) AMPure XP beads (vortexed or pipetted until homogeneous) to each well of the fourth well plate 126.
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes. After shaking the fourth well plate 126, liquid-handling system 100 allows the fourth well plate 126 to incubate at room temperature for 5 minutes. Then, one or more robotic arms 124 once again position the fourth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds. Next, one or more robotic arms 124 position the fourth well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the fourth well plate 126 to sit on the magnetic stand 138 for about 5 to 7 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then remove and discard the supernatant from each well (e.g., about 34.5 microliters of supernatant from each well).
  • liquid-handling system 100 After removing the supernatant from each well, and while the fourth well plate 126 continues to sit on the magnetic stand 138, liquid-handling system 100 “washes” the contents of each well by adding 30 microliters of 80% EtOH to each well using the one or more pipette devices 122, allowing the fourth well plate 126 to incubate on the magnetic stand 138 for 30 seconds after adding the 80% EtOH, and then removing and discarding supernatant from each well using the one or more pipette devices 122 (e.g., about 30 microliters of supernatant from each well). The liquid-handling system performs this wash twice. After the second wash, one or more pipette devices 122 remove residual EtOH from each well. Once the residual EtOH is removed, liquid-handling system 100 allows the contents of the wells to air-dry for 5 minutes (while the fourth well plate 126 continues to sit on the magnetic stand 138).
  • one or more robotic arms 124 remove the fourth well plate 126 from the magnetic stand 138 (e.g., and position the fourth well plate 126 on deck 118).
  • One or more pipette devices 122 then add 13.125 microliters of room temperature RSB to each well (with the AMPure XP beads still contained in each well).
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes.
  • one or more robotic arms 124 remove the fourth well plate 126 from orbital shaker 108 and position the fourth well plate 126 on centrifuge 106.
  • centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds. After centrifuge 106 centrifuges the fourth well plate 126, liquid-handling system 100 allows the fourth well plate 126 to incubate at room temperature for 2 minutes (e.g., while positioned on centrifuge 106 or while positioned on deck 118).
  • one or more pipette devices 122 add 12.5 microliters of 2.5 M Sodium Chloride, 20% PEG solution to each well of the fourth well plate 126 while the AMPure XP beads that were added to the wells earlier in step 212 (for the first cDNA fragment cleanup) are still contained in the wells.
  • Existing cDNA library generation protocols typically add SPRI paramagnetic beads (e.g., AMPure XP Beads) for this sub-step (the second cDNA fragment cleanup) instead of a PEG solution.
  • SPRI paramagnetic beads e.g., AMPure XP Beads
  • adding a PEG solution instead of paramagnetic beads, as described above, can be much more time efficient because SPRI paramagnetic beads take time to prepare.
  • SPRI paramagnetic beads need to be thoroughly mixed (e.g., vortexed or pipetted) for several minutes before use, whereas the 2.5 M Sodium Chloride, 20% PEG solution requires little to no preparation before being added to the wells of the fourth well plate 126.
  • adding the PEG solution for this sub-step can provide the additional benefit of increasing the final cDNA concertation (e.g., nanograms per microliter) of the wells (i.e., the final cDNA concertation after step 214).
  • adding the PEG solution to the wells while AMPure XP beads were contained within the wells increased the concertation of amplified/enriched cDNA per well by about 18 % when compared to the per-well cDNA concentration of existing cDNA library generation protocols that only use paramagnetic beads for ligated cDNA fragment cleanup (and do not utilize a PEG solution).
  • Using the PEG solution instead of SPRI paramagnetic beads for ligated cDNA fragment cleanup can also be more cost efficient because SPRI paramagnetic beads are much more expensive than the PEG solution.
  • one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes. After shaking the fourth well plate 126, liquid-handling system 100 allows the fourth well plate 126 to incubate at room temperature for 5 minutes. Then, one or more robotic arms 124 position the fourth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds. Next, one or more robotic arms 124 position the fourth well plate 126 on a magnetic stand 138.
  • Liquid-handling system then allows the fourth well plate 126 to sit on the magnetic stand 138 for about 5 to 7 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then remove and discard the supernatant from each well (e.g., about 34.5 microliters of supernatant from each well).
  • liquid-handling system 100 After removing the supernatant from each well, and while the fourth well plate 126 continues to sit on the magnetic stand 138, liquid-handling system 100 “washes” the contents of each well by adding 30 microliters of 80% EtOH to each well using one or more pipette devices 122, allowing the fourth well plate 126 to incubate on the magnetic stand 138 for 30 seconds after adding the 80% EtOH, and then removing and discarding supernatant from each well using the one or more pipette devices 122 (e.g., about 30 microliters of supernatant from each well). Liquid-handling system 100 performs this wash twice. After the second wash, one or more pipette devices 122 remove residual EtOH from each well. Once the residual EtOH is removed, liquid-handling system 100 allows the contents of the wells to air-dry for 5 minutes (while the fourth well plate 126 continues to sit on the magnetic stand 138).
  • one or more robotic arms 124 remove the fourth well plate 126 from the magnetic stand 138 (e.g., and position the fourth well plate 126 on deck 118). Then, one or more pipette devices 122 add 5.625 microliters of room temperature RSB to each well. Next, one or more robotic arms 124 position the fourth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fourth well plate 126 at 4000 rpm for 2 minutes. After, one or more robotic arms 124 remove the fourth well plate 126 from orbital shaker 108 and position the fourth well plate 126 on centrifuge 106.
  • centrifuge 106 centrifuges the fourth well plate 126 at 168 times gravity for 10 seconds. After centrifuge 106 centrifuges the fourth well plate 126, liquid-handling system 100 allows the fourth well plate 126 to incubate at room temperature for 2 minutes (e.g., while positioned on centrifuge 106 or while positioned on deck 118).
  • one or more robotic arms 124 position the fourth well plate 126 on magnetic stand 138.
  • Liquid-handling system 100 then allows the fourth well plate 126 to sit on the magnetic stand 138 for about 5 to 7 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then transfer 5 microliters of supernatant from each well of the fourth well plate 126 to corresponding wells of a fifth, unused well plate 126 (e.g., a 384- well PCR plate or 384-well MIDI plate).
  • one or more (e.g., each) of the wells of the fifth well plate 126 will contain double-stranded cDNA with RNA index adapters ligated to their ends (i.e., ligated onto the 3’ and 5’ end of both the first and second strands of the double-stranded cDNA).
  • step 212 takes about 229 minutes or less to complete.
  • one or more wells e.g., one or more wells that originally contained cells of a same cell line (e.g., human glioblastoma cells)
  • a same cell line e.g., human glioblastoma cells
  • liquid-handling system 100 amplifies the cDNA contained in the wells of the fifth well plate 126 via polymerase chain reaction (“PCR”) amplification, according to the following process.
  • PCR polymerase chain reaction
  • one or more robotic arms 124 position the fifth well plate 126 on icel 16 (or in some examples, a similarly-cold surface).
  • one or more pipette devices 122 add 7.5 microliters of a PCR Reagent mixture to each well of the fifth well plate 126.
  • the PCR Reagent mixture added to each well is taken from a source (e.g., a tube 132 or trough 136) containing a 5 to 1 mixture of previously-centrifuged (centrifuged at 168 times gravity for 10 seconds) PCR Master Mix (“PMM”) and previously-centrifuged (centrifuged at 168 times gravity for 10 seconds) PCR Primer Cocktail (“PPC”) (e.g., 5 microliters of PMM for every 1 microliter of PPC).
  • a source e.g., a tube 132 or trough 136
  • PMM PCR Master Mix
  • PPC PCR Primer Cocktail
  • the previously-centrifuged PMM and PCR are mixed together using one or more intermediate plates 128 during one or more of the previous sub- steps of step 212 (e.g., while allowing the fifth well plate 126 to sit on the magnetic stand 138 for 5 to 7 minutes).
  • intermediate plates to mix reagents as described above can expedite steps of process 200 and thus make the entire cDNA library generation more time efficient.
  • Existing cDNA library generation protocols typically add the PPC and PMM at two separate steps. Specifically, existing protocols first add PPC to wells and then pipette the contents of the wells several times in order to mix in the PPC and the contents of the wells. Then, once the PPC and the contents of the wells are thoroughly mixed, PMM is added to the wells. However, adding a mixture of PPC and PMM (i.e., the PCR Reagent mixture described above) to the wells of the fifth well plate 126 (instead of adding PPC and PMM separately) can be much more time efficient because it bypasses the additional, time consuming step of having to pipette the contents of each well after the PPC is added.
  • a mixture of PPC and PMM i.e., the PCR Reagent mixture described above
  • step 214 For example, on average, adding the PCR Reagent mixture to the wells of the fifth well plate 126 (instead of adding PPC, mixing thoroughly, and then adding PMM) makes this sub-step of step 214 about 45 minutes faster than that of existing cDNA library generation protocols.
  • one or more robotic arms 124 position the fifth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the fifth well plate 126 at 4000 rpm for 2 minutes. Then, after shaking the fifth well plate 126, one or more robotic arms 124 position the fifth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fifth well plate 126 at 168 times gravity for 10 seconds. Next, one or more robotic arms 124 position the fifth well plate 126 on thermal cycler 112 (which has had its lid preheated to 100 degrees Celsius).
  • the fifth well plate 126 undergoes the following pre-programmed thermal cycle (e.g., stored on memory 142): 98 degrees Celsius for 30 seconds, followed by 15 cycles of 98 degrees Celsius for 10 seconds, 60 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds, followed by 72 degrees Celsius for 5 minutes, and lastly followed by a constant 4 degrees Celsius until one or more robotic arms 124 remove the 384-well plate from thermal cycler 112.
  • pre-programmed thermal cycle e.g., stored on memory 142
  • one or more robotic arms 124 position the fifth well plate 126 on vortex mixer 110 and vortex mixer 110 vortexes the fifth well plate 126 for 5 seconds. After the fifth well plate 126 is vortexed for 5 seconds, one or more robotic arms 124 position the fifth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the fifth well plate 126 at 280 times gravity for 10 seconds.
  • one or more robotic arms 124 remove the fifth well plate 126 from centrifuge 106 (e.g., and position the fifth well plate 126 on deck 118) and then one or more pipette devices 122 add 12.5 microliters of previously-vortexed (or previously pipetted) AMPure XP beads (vortexed or pipetted until homogeneous) to each well of a sixth, unused well plate 126 (a 384-well PCR plate or 384-well MIDI plate).
  • one or more pipette devices 122 add another type of SPRI paramagnetic bead (e.g., CleanNGS SPRI Beads) to the sixth well plate 126 instead of AMPure XP beads.
  • the liquid handling system transfers 12.5 microliters of the contents of each well of the fifth well plate 126 to corresponding wells of the sixth well plate 126 (which already contain 12.5 microliters of AMPure XP beads).
  • one or more robotic arms 124 position the sixth well plate 126 on orbital shaker 108 and orbital shaker 108 shakes the sixth well plate 126 at 4000 rpm for 2 minutes. After shaking the sixth well plate 126, liquid-handling system 100 allows the sixth well plate 126 to incubate at room temperature for 15 minutes. One or more robotics arms 124 then position the sixth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the sixth well plate 126 at 168 times gravity for 10 seconds. Next, one or more robotic arms 124 position the sixth well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the sixth well plate 126 to sit on the magnetic stand 138 for about 5 to 7 minutes (or until the liquid content of the wells is clear).
  • One or more pipette devices 122 then remove and discard the supernatant from each well (e.g., about 34.5 microliters of supernatant from each well).
  • liquid-handling system 100 After removing the supernatant from each well, and while the sixth well plate 126 continues to sit on the magnetic stand 138, liquid-handling system 100 “washes” the contents of each well by adding 30 microliters of 80% EtOH to each well using one or more pipette devices 122, allowing the sixth well plate 126 to incubate on the magnetic stand 138 for 30 seconds after adding the 80% EtOH, and then removing and discarding supernatant from each well using one or more pipette devices 122 (e.g., about 30 microliters of supernatant from each well). Liquid-handling system 100 performs this wash twice. After the second wash, one or more pipette devices 122 remove residual EtOH from each well.
  • liquid-handling system 100 allows the contents of the wells to air-dry for 5 minutes (while the 384-well plate continues to sit on the magnetic stand).
  • one or more robotic arms 124 remove the sixth well plate 126 from the magnetic stand 138 (e.g., and position the sixth well plate 126 on deck 118).
  • One or more pipette devices 122 then add 20 microliters of room temperature RSB to each well.
  • one or more robotic arms 124 position the sixth well plate 126 on orbital shaker 108 and orbital shaker shakes the sixth well plate 126 at 4000 rpm for 2 minutes.
  • Liquid-handling system 100 then allows the sixth well plate 126 to incubate at room temperature for 2 minutes.
  • one or more robotic arms 124 position the sixth well plate 126 on centrifuge 106 and centrifuge 106 centrifuges the sixth well plate 126 at 168 times gravity for 10 seconds.
  • one or more robotic arms 124 position the sixth well plate 126 on a magnetic stand 138.
  • Liquid-handling system 100 then allows the sixth well plate 126 to sit on the magnetic stand 138 for about 5 to 7 minutes (or until the liquid content of the wells is clear).
  • one or more pipette devices 122 transfer 20 microliters of supernatant from each well of the sixth well plate 126 to corresponding wells of an seventh, unused well plate 126 (a 384-well PCR plate). In some examples, one or more pipette devices 122 transfer more (e.g., 25 microliters) or less (e.g., 18 microliters) of supernatant from each well of the sixth well plate 126 to the corresponding wells of the seventh well plate 126.
  • step 214 takes about 53 minutes or less to complete.
  • process 200 only takes about 995 minutes or less to complete, while producing an average concentration of 5 nanograms of enriched cDNA per microliter of well contents at the end of step 214 (or about 100 nanograms of enriched cDNA per well).
  • Process 200 can be significantly more time efficient than existing cDNA library generation protocols, as existing protocols require at least 4,600 minutes on average to produce amplified/enriched cDNA for an equivalent number of samples/wells.
  • process 200 can also be much more cost efficient per sample than existing cDNA library generation protocols for at least the reason that process 200 uses much lower volumes of reagents (which can be very expensive) for each of its steps when compared to equivalent steps of existing cDNA library generation protocols.
  • existing cDNA library generation protocols cost approximately four times more per sample when compared to the per sample cost of process 200 (e.g., cost of disposable materials, reagents, etc.).
  • process 200 is performed using well plates 126 that have more or less than 384 wells (although this will increase or decrease the total volume of each reagent that is used and the amount of time each step takes).
  • one or more steps of process 200 can be performed using 96-well plates and/or 192-well plates instead of 384- well plates (e.g., 96-well MIDI plates).
  • alternative reagents may be used for one or more steps of process 200.
  • Example alternative reagents include NEBNext® Ultra II RNA Library Prep reagents (New England Biolabs®) and KRAP RNA Library Preparation kit reagents (Roche®).
  • one or more operations of process 200 described above as being performed by liquid-handling system 100 are instead performed by one or more operators of liquid-handling system 100.
  • one or more operators of liquid-handling system 100 remove the well plates 126 from automated pipetting system 102 (e.g., from deck 118) and position the well plates 126 on a separately-located orbital shaker 108 (e.g., located next to automated pipetting system 102).
  • FIG. 3 illustrates a flow chart representing an exemplary process for RNA sequence amplification.
  • Process 300 is merely exemplary. Thus, some operations in process 300 are, optionally, combined, the orders of some operations are, optionally, changed, and some operations are, optionally, omitted. In some examples, one or more steps of process 300 are performed by a system similar or identical to liquid-handling system 100, described above with reference to FIG. 1.
  • a sample well plate within a liquid handling system (e.g., an automated liquid-handling system (e.g., Hamilton Microlab STAR Liquid Handling System)), wherein the sample well plate includes a plurality of wells containing cells (e.g., human cells and/or animal cells).
  • a liquid handling system e.g., an automated liquid-handling system (e.g., Hamilton Microlab STAR Liquid Handling System)
  • the sample well plate includes more than 96 wells (e.g., 384 wells).
  • a plurality of the wells included in the sample well plate each contain a different set of cells (e.g., cells of different cell lines (e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like) and/or cells from different sources (e.g., human and/or animal cells)).
  • a different set of cells e.g., cells of different cell lines (e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like) and/or cells from different sources (e.g., human and/or animal cells).
  • the sample well plate includes more than 96 wells, at least 96 of those wells each contain a different set of cells.
  • step 304 produce cell lysate (e.g., contents of one or more lysed cells (e.g., lysed human cells and/or animal cells)) in at least a first well of the plurality of wells using the cells contained in at least the first well, wherein the cell lysate includes mRNA (e.g., as described above with reference to step 202).
  • the cell lysate is a whole cell lysate.
  • the cell lysate further includes DNA.
  • producing the cell lysate further includes adding a DNase reagent to at least the first well to digest DNA included in the cell lysate.
  • the cell lysate produced in the first well is produced from a first set of cells (e.g., cells of a first cell line (e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like)), and the cell lysate produced in a second well of the plurality of wells is produced from a second set of cells different from the first set of cells (e.g., cells of a second cell line (e.g., adenocarcinoma cells, melanoma cells, glioblastoma cells, or the like)).
  • producing the cell lysate takes no more than 90 minutes.
  • mRNA e.g., separate mRNA from the rest of the cell lysate contents, including other forms of RNA (e.g., ribosomal RNA)
  • purifying the mRNA includes adding RNA purification beads to at least the first well and mixing contents of the plurality of wells via planar orbital motion (e.g., using a high-speed sample well plate shaker (e.g., Bioshake 5000)).
  • step 308 fragment the purified mRNA in at least the first well into a plurality of mRNA fragments (e.g., as described above with reference to step 204).
  • step 310 prime at least one mRNA fragment of the plurality of mRNA fragments in at least the first well (e.g., for cDNA synthesis (e.g., using random hexamer primers)) (e.g., as described above with reference to step 204), wherein the cDNA is synthesized in at least the first well (at step 312) using the at least one primed mRNA fragment.
  • cDNA synthesis e.g., using random hexamer primers
  • step 312 synthesize cDNA in at least the first well (e.g., single-stranded and double-stranded cDNA synthesis) using the purified mRNA (e.g., as described above with reference to step 206 and/or step 208).
  • the purified mRNA e.g., as described above with reference to step 206 and/or step 208.
  • synthesizing the cDNA in at least the first well includes adding a mixture of a first strand synthesis reagent (e.g., First Strand Synthesis Act D Mix) and a reverse transcriptase reagent (e.g., Superscript III Reverse Transcriptase) to at least first well, wherein the first strand synthesis reagent and the reverse transcriptase reagent are mixed together using an intermediate well plate of the liquid handling system (e.g., separate from the sample well plate) prior to being added to at least the first well.
  • a first strand synthesis reagent e.g., First Strand Synthesis Act D Mix
  • a reverse transcriptase reagent e.g., Superscript III Reverse Transcriptase
  • the first strand synthesis reagent and the reverse transcriptase reagent are mixed together using the intermediate plate during a preceding step of the method (e.g., while purifying the mRNA or while fragmenting the purified mRNA).
  • a total volume of the mixture of the first strand synthesis reagent and the reverse transcriptase reagent added to the first well is equal to or less than 2.0 microliters (e.g., equal to or less than 1 microliter for every 12.5 microliters of cell lysate initially contained in the first well).
  • purifying the mRNA, fragmenting the purified mRNA, priming the fragmented mRNA, and synthesizing the cDNA takes no more than 515 minutes to complete.
  • preparing the synthesized cDNA in at least the first well for amplification includes adenylating (e.g., adding one adenine (A) nucleotide to) a 3’ end of a first strand of the synthesized cDNA and a 3’ end of a second strand of the synthesized cDNA (e.g., as described above with reference to step 210) and ligating (e.g., attaching) an index adapter (e.g., a single-index adapter) to each end (e.g., the 3’ end and a 5’end) of the first strand cDNA and to each end of the second strand cDNA (e.g., as described above with reference to step 210 (e.g., ligating an index adapter to the ends
  • an index adapter e.g., a single-index adapter
  • ligating the index adapter includes adding a ligation reagent to at least the first well, cooling contents of at least the first well after adding the ligation reagent, and adding a stop ligation buffer to at least the first well while cooling the contents of at least the first well.
  • a total volume of the stop ligation buffer added to the first well is equal to or less than 1.25 microliters (e.g., equal to or less than 1 microliter for every 20 microliters of cell lysate initially contained in the first well).
  • preparing the synthesized cDNA in at least the first well for amplification includes adding solid-phase reversible immobilization (SPRI) paramagnetic beads (e.g., AMPure XP beads) to at least the first well, and while the SPRI paramagnetic beads are in at least the first well, adding a polyethylene glycol (PEG) reagent to at least the first well.
  • SPRI solid-phase reversible immobilization
  • PEG polyethylene glycol
  • the SPRI paramagnetic beads purify contents of at least the first well (e.g., assist with removing cDNA fragments that do not have ligated index adapters).
  • the PEG reagent further purifies the contents of at least the first well.
  • amplify the prepared cDNA in at least the first well (e.g., as described above with reference to step 214). Amplifying the prepared cDNA in at least the first well increases an amount (e.g., a number of strands) of cDNA in at least the first well.
  • amplifying the prepared cDNA in at least the first well includes adding a polymerase chain reaction (PCR) reagent (e.g., PCR Master Mix) to the well.
  • PCR polymerase chain reaction
  • a total volume of the PCR reagent added to the first well is equal to or less than 6.25 microliters (e.g., equal to or less than 1 microliters for every 4 microliters of cell lysate initially contained in the first well).
  • amplifying the prepared cDNA in at least the first well further includes adding a PCR primer reagent (e.g., PCR Primer Cocktail) to at least the first well when adding the PCR reagent to at least the first well.
  • the PCR primer reagent and the PCR reagent are mixed together using an intermediate well plate prior to being added to at least the first well.
  • a total volume of the PCR primer reagent added to the first well is equal to or less than 1.25 microliters (e.g., equal to or less than 1 microliter for every 20 microliters of cell lysate initially contained in the first well).
  • preparing the synthesized cDNA and amplifying the prepared cDNA takes no more than 395 minutes to complete.
  • the liquid-handling system is an automated liquid-handling system.
  • producing the cell lysate, purifying the mRNA, synthesizing the cDNA, preparing the synthesized cDNA, and amplifying the prepared cDNA are automatically performed by the liquid handling system.
  • the cell lysate in the first well includes no more than 1 nanogram of mRNA (e.g., or no more than 10 nanograms of total RNA (which is composed of 1-5% of mRNA)), and process 300 produces a concentration of at least 5 nanograms of cDNA per microliter of liquid contents (e.g., elution) in the first well in the first well (e.g.,
  • process 300 takes less than 1000 minutes to complete after the generation of the cell lysate in at least the first well begins.

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Abstract

La présente divulgation concerne un protocole amélioré de génération de banque d'ADNc pour générer une banque d'ADN complémentaire ("ADNc") en moins de temps et à moindre coût que les protocoles existants de génération de banque d'ADNc. Un exemple de procédé comprend les étapes suivantes : positionnement d'une plaque de puits d'échantillon à l'intérieur d'un système de manipulation de liquide, la plaque de puits d'échantillon comprenant une pluralité de puits contenant des cellules ; production d'un lysat cellulaire dans au moins un premier puits de la pluralité de puits en utilisant les cellules contenues dans au moins le premier puits, dans lequel le lysat cellulaire comprend un ARN messager (ARNm) ; purification de l'ARNm dans au moins le premier puits ; synthèse d'un ADNc dans au moins le premier puits en utilisant l'ARNm purifié ; préparation de l'ADNc synthétisé dans au moins le premier puits pour l'amplification ; et amplification de l'ADNc préparé dans au moins le premier puits, dans lequel l'amplification de l'ADNc préparé augmente une quantité d'ADNc dans au moins le premier puits.
PCT/US2021/030895 2020-05-22 2021-05-05 Génération de banque d'adnc WO2021236328A1 (fr)

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WO2017013103A1 (fr) * 2015-07-23 2017-01-26 Biocartis Nv Échantillon automatisé pour la préparation d'une bibliothèque de séquençage de nouvelle génération
WO2018057961A1 (fr) * 2016-09-23 2018-03-29 ArcherDX, Inc. Ensemble magnétique
WO2020023744A1 (fr) * 2018-07-26 2020-01-30 Lexent Bio, Inc. Séquençage multiple à l'aide d'une cellule à flux unique

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