JP2017522867A - 個々の細胞または細胞集団由来の核酸の分析方法 - Google Patents
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Abstract
Description
本出願は、2014年6月26日出願の米国仮特許出願番号第62/017,558号及び2014年10月8日出願の米国仮特許出願番号第62/061,567号の優先権を主張し、それらの出願のそれぞれは、全ての目的のために参照によりその全体が本明細書に組込まれる。
本明細書で言及される全ての刊行物、特許、及び特許出願は、個々の刊行物、特許、または特許出願が、具体的にかつ個別に参照により組込まれているかのように示されているのと同じ程度で、参照により本明細書に組込まれる。参照により組込まれる刊行物及び特許または特許出願が、本明細書に含まれる開示と矛盾する範囲で、本明細書は、そのような任意の矛盾する材料に代わるかつ/または優先することを意図している。
先進的な核酸配列決定技術は、個々の生物及び比較的純粋な生物学的サンプルの実質的な配列情報を提供することを含む、生物学的材料を配列決定することにおける驚異的な結果をもたらしている。しかし、これらのシステムは、サンプルの全体的な構成のごく一部を表し得る、かつ個々の配列情報がさらに有益なものとなり得る生物学的サンプル中の細胞のサブ集団を識別して特徴付けることにおいて有効であるとは証明されていない。
しかし、本明細書で開示されるのは、小さな細胞の集団から、かつ、いくつかの場合には、特に細胞の集団がより大きい状況において、個々の細胞からの核酸を特徴付けるための方法及びシステムである。この方法及びシステムは、個々の細胞または細胞の小集合から抽出可能な非常に少量の入力核酸を処理して配列決定することができるというさらなる利点とともに、他の次世代システムのスループットが高い非増幅単一分子法の帰属の利点を提供する。
特定の個々のエル(ells)の分析、異なる細胞タイプの集団内の異なる細胞タイプの分析、環境、ヒトの健康、疫学法医学に関する、細胞の大集団の分析及び特徴付けを含む、本明細書に記載の単一細胞処理及び分析方法及びシステムの多様な応用が存在する。
本明細書では、上記のように細胞を区分化するのに使用されるマイクロ流体装置も提供される。このようなマイクロ流体装置は、図1及び2に示されるような区分化プロセスを実行するためのチャンネルネットワークを含むことができる。特に有用なマイクロ流体装置の例は、2014年4月4日に出願された米国仮特許出願第61/977,804号に記載されており、全ての目的のためにその全体が参照により本明細書に組込まれる。簡潔には、これらのマイクロ流体装置は、細胞を別個の細胞に区分化させるために、かつそのような細胞を、例えば、ビーズ上に配置されたオリゴヌクレオチドバーコードライブラリーメンバーと共に区分化させるために、本明細書に記載されているものなどのチャンネルネットワークを備えることができる。これらのチャンネルネットワークは、固体ボディ、例えば、チャンネルが画定されるガラス、半導体またはポリマーのボディ構造内に配置することができるが、この場合、それらのチャンネルは、その端部で、例えば、チャンネルネットワークの出力から、様々な入力流体を受容するために、かつ区分化された細胞を最終的に堆積させるために、端部で容器と連通する。例として、図2を参照すると、チャンネル202と流体連結する容器には、細胞214の水性懸濁液が提供されてもよく、チャンネル204に連結する容器には、オリゴヌクレオチドを担持するビーズ216の水性懸濁液が提供されてもよい。チャンネルセグメント206及び208には、非水性溶液、例えば油が供給されてもよく、この中に水性流体がチャンネル接合部212において液滴として区分化される。最終的に、排出容器を、チャンネル210に流体連結させて、そこに区分化された細胞及びビーズを送達することができ、かつそこからそれらを採取してもよい。理解されるように、容器として記載されているが、チャンネルセグメントは、他のシステムの配管、マニホールド、または流体構成要素を含む様々な異なる流体源または受容構成要素のいずれかに連結され得ることが理解されるであろう。
本明細書では、個々の細胞または細胞の小さな集団を分析するためのキットも提供される。キットには、水性緩衝液及び非水性区分化流体または油の両方を含む、最大1つ、2つ、3つ、4つ、5つ、またはそれ以上の全ての区分化流体、本明細書に記載のようにビーズと放出可能に結合している核酸バーコードライブラリー、マイクロ流体装置、細胞を破壊し、核酸を増幅させ、かつ細胞の核酸の断片またまたはその複製物にさらなる機能配列を提供するための試薬、ならびに本明細書に記載の方法で上述のいずれかのものを使用するための説明書が含まれ得る。
本開示は、本開示の方法を実施するようにプログラムされているコンピュータ制御システムを提供する。図17は、核酸配列決定方法、核酸配列データの解釈及び細胞核酸、RNA(例えば、mRNA)などの分析、ならびに配列データから細胞を特徴付けることを含む、本開示の方法を実施するようにプログラムされているかまたはさもなければ構成されている、コンピュータシステム1701を示す。コンピュータシステム1701は、電子デバイスに対して遠隔に配置されたユーザーまたはコンピュータシステムの電子デバイスであってよい。電子デバイスは、モバイル電子デバイスであってよい。
実施例I エマルジョンを用いた細胞RNA分析
一例では、図9Aに示す操作で、エマルジョン液滴中で、テンプレートスイッチングによる逆転写及びcDNA増幅(PCRによるもの)を行う。逆転写及びcDNA増幅(PCRによるもの)のために区分化された反応混合物は、1,000の細胞もしくは10,000の細胞または10ngのRNA、バーコード化オリゴヌクレオチド/0.2%Tx−100/5倍のKapa緩衝液を含むビーズ、2倍のKapa HS HiFi Ready Mix、4μMのスイッチオリゴ、ならびにSmartscribeを含む。細胞が存在する場合、混合物は、大部分または全ての液滴が単一細胞及び単一ビーズを含むように区分化される。操作950のように、細胞が溶解され、その間バーコード化オリゴヌクレオチドがビーズから放出され、バーコード化オリゴヌクレオチドのポリTセグメントが、細胞から放出されたmRNAのポリA尾部にハイブリダイズする。ポリTセグメントは、操作952のように逆転写反応で伸長され、cDNA転写物は操作954のように増幅される。熱サイクル条件は42℃で130分間、98℃で2分間、ならびに98℃で15秒間、60℃で20秒間、及び72℃で6分間の35サイクルである。熱サイクル後、エマルジョンは破壊され、操作956と同様に転写物はDynabeads及び0.6倍のSPRIで精製される。
別の例では、図9Aに示す操作で、エマルジョン液滴中で、テンプレートスイッチングによる逆転写及びcDNA増幅(PCRによるもの)を行う。逆転写及びcDNA増幅(PCRによるもの)のために区分化された反応混合物は、Jurkat細胞、バーコード化オリゴヌクレオチド/0.2% TritonX−100/5倍のKapa緩衝液を含むビーズ、2倍のKapa HS HiFi Ready Mix、4μMのスイッチオリゴ、及びSmartscribeを含む。混合物は、大部分または全ての液滴が単一細胞及び単一ビーズを含むように区分化される。操作950のように、細胞が溶解され、その間バーコード化されたオリゴヌクレオチドがビーズから放出され、バーコード化されたオリゴヌクレオチドのポリTセグメントが、細胞から放出されたmRNAのポリA尾部にハイブリダイズする。ポリTセグメントは、操作952のように逆転写反応で伸長され、cDNA転写物は操作954のように増幅される。熱サイクル条件は42℃で130分間、98℃で2分間、ならびに98℃で15秒間、60℃で20秒間、及び72℃で6分間の35サイクル、である。熱サイクル後、エマルジョンは破壊され、操作956と同様に転写物はDynabeads及び0.6倍のSPRIで洗浄される。様々な細胞数(625の細胞、1,250の細胞、2,500の細胞、5,000の細胞、10,000の細胞)との反応からの収率を図14Aに示す。これらの収率は、図14Bに示されるGADPH qPCRアッセイ結果で確認される。
別の例では、図9Cに示されるのと同様の方法で、エマルジョン液滴中で逆転写を行い、バルクでcDNA増幅を行う。逆転写反応のために区分化される反応混合物は、バーコード化されたオリゴヌクレオチドを有するビーズ、10ngのJurkat RNA(例えばJurkat mRNA)、5倍のFirst−Strand緩衝液、及びSmartscribeを含む。バーコード化オリゴヌクレオチドはビーズから放出され、バーコード化オリゴヌクレオチドのポリTセグメントは、操作961のようにRNAのポリA尾部にハイブリダイズする。ポリTセグメントは、操作963のように逆転写反応において伸長される。逆転写の熱サイクル条件は、42℃で2時間の1サイクル、及び70℃で10分間の1サイクルである。熱サイクル後、エマルジョンは破壊され、操作962のようにRNA及びcDNA転写物は変性される。次いで、第2鎖は、操作964のようにビオチンタグを有するプライマーでプライマー伸長により合成する。このプライマー伸長の反応条件は、第1鎖としてのcDNA転写物、及び0.5〜3.0μMの濃度範囲のビオチン化伸長プライマーを含む。熱サイクル条件は、98℃で3分間の1サイクル、ならびに98℃で15秒間、60℃で20秒間、及び72℃で30分間の1サイクルである。プライマーの伸長後、第2鎖をDynabeads MyOne Streptavidin C1及びT1でプルダウンし、Agilent SureSelect XT緩衝液で洗浄する。第2鎖は、以下のサイクル条件、すなわち98℃で3分間の1サイクル、ならびに98℃で15秒間、60℃で20秒間、及び72℃で30分間の1サイクルで、操作965のようにPCRにより事前に増幅される。様々な濃度のビオチン化プライマー(0.5μM、1.0μM、2.0μM及び3.0μM)の収率を図15に示す。
別の例では、図10に示すように、T7ポリメラーゼによるインビトロ転写がRNA転写物を生成するのに使用される。逆転写反応のために区分化される反応混合物は、T7RNAポリメラーゼプロモーター配列も含むバーコード化されたオリゴヌクレオチドを有するビーズ、10ngのヒトRNA(例えば、ヒトmRNA)、5倍のFirst−Strand緩衝液、及びSmartscribeを含む。混合物は、液滴の大部分または全部が単一のビーズを含むように区分化される。バーコード化オリゴヌクレオチドはビーズから放出され、バーコード化オリゴヌクレオチドのポリTセグメントは、操作1050のようにRNAのポリA尾部にハイブリダイズする。ポリTセグメントは、操作1052のように逆転写反応において伸長される。熱サイクル条件は、42℃で2時間の1サイクル及び70℃10分間で1サイクルである。熱サイクル後、エマルジョンは破壊され、残りの操作はバルクで行われる。次いで、第2鎖が、操作1054のようにプライマー伸長によって合成される。このプライマー伸長の反応条件は、テンプレートとしてのcDNA転写物及び伸長プライマーを含む。熱サイクル条件は、98℃で3分間の1サイクル、ならびに98℃で15秒間、60℃で20秒間、及び72℃30分間の1サイクルである。このプライマー伸長後、第2鎖を0.6倍のSPRIで精製する。次いで、操作1056のように、RNA転写物を生成するためにインビトロ転写が行われる。インビトロ転写を一晩行い、転写物を0.6倍のSPRIで精製する。インビトロ転写からのRNA収率を図16に示す。
Claims (88)
- 細胞から核酸を分析する方法であって、
(a)個々の細胞に由来する核酸を離散区分に提供することと、
(b)前記離散区分内の前記核酸に由来する1つまたは複数の第1の核酸配列であって、共通の核酸バーコード配列を含むオリゴヌクレオチドを結合させている前記1つまたは複数の第1の核酸配列を生成することと、
(c)前記1つもしくは複数の第1の核酸配列または前記1つもしくは複数の第1の核酸配列に由来する1つもしくは複数の第2の核酸配列であって、共通のバーコード配列を含む前記1つもしくは複数の第2の核酸配列の特徴付けを行うことと、
(d)(c)で行われた前記特徴付けにおける前記共通の核酸バーコード配列の存在に少なくとも部分的に基づいて、前記個々の細胞に由来する前記1つもしくは複数の第1の核酸配列または1つもしくは複数の第2の核酸配列を識別することと、を含む、前記方法。 - 前記離散区分が離散液滴である、請求項1に記載の方法。
- (a)において、前記オリゴヌクレオチドが、前記個々の細胞に由来する前記核酸と前記離散区分に共に区分される、請求項1に記載の方法。
- (a)において、少なくとも10,000の前記オリゴヌクレオチドが、前記離散区分に前記個々の細胞に由来する前記核酸と共に区分される、請求項3に記載の方法。
- (a)において、少なくとも100,000の前記オリゴヌクレオチドが、前記離散区分に前記個々の細胞に由来する前記核酸と共に区分される、請求項4に記載の方法。
- (a)において、少なくとも500,000の前記オリゴヌクレオチドが、前記離散区分に前記個々の細胞に由来する前記核酸と共に区分される、請求項5に記載の方法。
- (a)において、前記オリゴヌクレオチドがビーズに付着されて提供され、ビーズ上の各オリゴヌクレオチドが同じバーコード配列を含み、前記ビーズが前記離散区分に前記個々の細胞と共に区分される、請求項1に記載の方法。
- 前記オリゴヌクレオチドが前記ビーズに放出可能に付着している、請求項7に記載の方法。
- 前記ビーズが分解性ビーズを含む、請求項8に記載の方法。
- (b)の前または最中に、前記ビーズの分解を介して前記ビーズから前記オリゴヌクレオチドを放出することをさらに含む、請求項9に記載の方法。
- (c)の前に、前記離散区分から前記1つまたは複数の第1の核酸配列を放出することをさらに含む、請求項1に記載の方法。
- (c)が、前記1つもしくは複数の第1の核酸配列または前記1つもしくは複数の第2の核酸配列を配列決定することを含む、請求項1に記載の方法。
- 前記1つもしくは複数の第1の核酸配列または前記1つもしくは複数の第2の核酸配列から、前記個々の細胞のゲノムの少なくとも一部の連続核酸配列をアセンブルすることをさらに含む、請求項12に記載の方法。
- 前記個々の細胞が、前記個々の細胞の前記ゲノムの少なくとも一部の前記核酸配列に基づいて特徴付けされる、請求項13に記載の方法。
- 前記核酸が、前記離散区分内の前記個々の細胞から放出される、請求項1に記載の方法。
- 前記核酸がリボ核酸(RNA)を含む、請求項1に記載の方法。
- 前記RNAがメッセンジャーRNA(mRNA)である、請求項16に記載の方法。
- (b)が、前記1つまたは複数の第1の核酸配列をもたらす条件下で前記核酸を逆転写させることをさらに含む、請求項16に記載の方法。
- 前記逆転写が前記離散区分で生じる、請求項18に記載の方法。
- 前記オリゴヌクレオチドが、前記離散区分に提供され、かつポリT配列をさらに含む、請求項18に記載の方法。
- 前記逆転写が、前記ポリT配列を前記核酸のそれぞれの少なくとも一部にハイブリダイズさせることと、前記ポリT配列をテンプレート指向の様式で伸長させることと、を含む、請求項20に記載の方法。
- 前記オリゴヌクレオチドが、前記ポリT配列のハイブリダイゼーションを促進するアンカー配列をさらに含む、請求項21に記載の方法。
- 前記オリゴヌクレオチドが、ランダムプライミング配列をさらに含む、請求項20に記載の方法。
- 前記ランダムプライミング配列がランダム六量体である、請求項23に記載の方法。
- 前記逆転写が、前記ランダムプライミング配列を前記核酸のそれぞれの少なくとも一部にハイブリダイズさせることと、前記ランダムプライミング配列をテンプレート指向の様式で伸長させることと、を含む、請求項24に記載の方法。
- 前記1つまたは複数の第1の核酸配列のうちの所与の1つが、前記核酸の所与の1つの少なくとも一部に対する配列相補性を有する、請求項1に記載の方法。
- 前記離散区分が、複数の細胞の中で、最大でも前記個々の細胞のみを含む、請求項1に記載の方法。
- 前記オリゴヌクレオチドが、固有の分子配列セグメントをさらに含む、請求項1に記載の方法。
- 前記固有の分子配列セグメントの存在に少なくとも部分的に基づいて、前記核酸の所与の核酸に由来する前記1つもしくは複数の第1の核酸配列、または前記1つもしくは複数の第2の核酸配列の個々の核酸配列を識別することをさらに含む、請求項28に記載の方法。
- 前記固有の分子配列セグメントの存在に基づいて前記所与の核酸の量を決定することをさらに含む、請求項29に記載の方法。
- (c)の前に、前記1つまたは複数の第2の配列を生成するために、1つまたは複数の追加配列を前記1つまたは複数の第1の配列に追加することをさらに含む、請求項1に記載の方法。
- スイッチオリゴヌクレオチドを用いて、第1の追加核酸配列を前記1つまたは複数の第1の核酸配列に追加することをさらに含む、請求項31に記載の方法。
- 前記スイッチオリゴヌクレオチドが、前記1つまたは複数の第1の核酸配列の少なくとも一部にハイブリダイズさせ、前記第1の追加核酸配列を前記1つまたは複数の第1の核酸配列に結合させるためにテンプレート指向様式で伸長する、請求項32に記載の方法。
- 前記第1の追加核酸配列に結合する前記より多くの第1の核酸配列のうちの1つを増幅させることをさらに含む、請求項33に記載の方法。
- 前記増幅が前記離散区分で生じる、請求項34に記載の方法。
- 前記増幅が、前記第1の追加核酸配列に結合する前記1つまたは複数の第1の核酸配列を前記離散区分から放出した後に生じる、請求項34に記載の方法。
- 前記増幅後に、前記1つまたは複数の第2の核酸配列を生成するために、前記第1の追加配列に結合する前記1つまたは複数の第1の核酸配列に1つまたは複数の第2の追加核酸配列を追加することをさらに含む、請求項34に記載の方法。
- 前記1つまたは複数の第2の追加配列の追加が、前記第1の追加核酸配列に結合する前記1つまたは複数の第1の核酸配列のそれぞれの一部を除去することと、前記1つまたは複数の第1の核酸配列に前記1つまたは複数の第2の追加核酸配列を結合させることと、を含む、請求項37に記載の方法。
- 前記除去が、前記第1の追加核酸配列に結合する前記1つまたは複数の第1の核酸配列の剪断によって完了する、請求項38に記載の方法。
- 前記結合が連結によって完了する、請求項39に記載の方法。
- (c)の前に、1つまたは複数のRNA断片を生成するために、前記1つまたは複数の第1の核酸配列を転写させることをさらに含む、請求項18に記載の方法。
- 前記転写が、離散区分からの前記1つまたは複数の第1の核酸配列の放出後に生じる、請求項41に記載の方法。
- 前記オリゴヌクレオチドが、T7プロモーター配列をさらに含む、請求項41に記載の方法。
- (c)の前に、前記1つまたは複数のRNA配列のそれぞれの一部を除去することと、前記1つまたは複数のRNA配列に追加配列を結合させることと、をさらに含む、請求項43に記載の方法。
- (c)の前に、前記1つまたは複数の第2の核酸配列を生成するために、前記追加配列に結合する前記1つまたは複数のRNA配列を逆転写させることをさらに含む、請求項44に記載の方法。
- (c)の前に、前記1つまたは複数の第2の核酸配列を増幅させることをさらに含む、請求項45に記載の方法。
- (c)の前に、1つまたは複数のDNA配列を生成するために、前記1つまたは複数のRNA配列を逆転写させることをさらに含む、請求項41に記載の方法。
- (c)の前に、前記1つまたは複数の第2の核酸配列を生成するために、前記1つまたは複数のDNA配列のそれぞれの一部を除去することと、前記1つまたは複数のDNA配列に1つまたは複数の追加配列を結合させることと、をさらに含む、請求項47に記載の方法。
- (c)の前に、前記1つまたは複数の第2の核酸配列を増幅させることをさらに含む、請求項48に記載の方法。
- 前記核酸が、前記個々の細胞からのRNAの逆転写から生成された相補的(cDNA)を含む、請求項1に記載の方法。
- 前記オリゴヌクレオチドがプライミング配列をさらに含む、離散区分に提供される、請求項50に記載の方法。
- 前記プライミング配列が、ランダムN量体を含む、請求項51に記載の方法。
- (b)が、前記ライミング配列を前記cDNAにハイブリダイズさせることと、前記プライミング配列をテンプレート指向の様式で伸長させることと、を含む、請求項51に記載の方法。
- 前記離散区分が、前記オリゴヌクレオチドの相補配列を含むスイッチオリゴヌクレオチドを含む、請求項1に記載の方法。
- (b)が、前記スイッチオリゴヌクレオチドを前記核酸由来の核酸断片の少なくとも一部にハイブリダイズさせることと、前記スイッチオリゴヌクレオチドをテンプレート指向の様式で伸長させることと、を含む、請求項54に記載の方法。
- (b)が、前記オリゴヌクレオチドを前記1つまたは複数の第1の核酸配列に結合させることを含む、請求項1に記載の方法。
- 前記1つまたは複数の第1の核酸配列が前記核酸由来の核酸断片である、請求項1に記載の方法。
- 前記(b)が、前記オリゴヌクレオチドを前記核酸に結合させることを含む、請求項1に記載の方法。
- 前記結合させることが連結させることを含む、請求項58に記載の方法。
- 複数の区分が離散区分を含む、請求項1に記載の方法。
- 前記複数の区分が、1つの区分あたり平均1つ未満の細胞を含む、請求項60に記載の方法。
- 前記複数の区分の25%未満の区分が細胞を含まない、請求項60に記載の方法。
- 前記複数の区分が、それぞれ少なくとも1つの区分された細胞を有する離散区分を含む、請求項60に記載の方法。
- 前記離散区分の25%未満が複数の細胞を含む、請求項63に記載の方法。
- 少なくとも離散区分のサブセットがビーズを含む、請求項64に記載の方法。
- 前記離散区分の少なくとも75%が、少なくとも1つの細胞及び少なくとも1つのビーズを含む、請求項65に記載の方法。
- 前記離散区分が、区分された核酸バーコード配列をさらに含む、請求項63に記載の方法。
- 前記離散区分が、少なくとも1,000の異なる区分された核酸バーコード配列を含む、請求項67に記載の方法。
- 前記離散区分が、少なくとも10,000の異なる区分された核酸バーコード配列を含む、請求項68に記載の方法。
- 前記離散区分が、少なくとも100,000の異なる区分された核酸バーコード配列を含む、請求項69に記載の方法。
- 前記複数の区分が、少なくとも1,000の区分を含む、請求項60に記載の方法。
- 前記複数の区分が少なくとも10,000の区分を含む、請求項71に記載の方法。
- 前記複数の区分が、少なくとも100,000の区分を含む、請求項72に記載の方法。
- 複数の異なる細胞タイプの集団における細胞を特徴付ける方法であって、
(a)前記集団内の個々の細胞からの核酸を離散区分へ提供することと、
(b)共通の核酸バーコード配列を含むオリゴヌクレオチドを、前記離散区分であって、複数の異なる区分が異なる共通の核酸バーコード配列を含む、前記離散区分内の個々の細胞からの前記核酸の1つまたは複数の断片に結合させることと、
(c)前記複数の離散区分からの前記核酸の前記1つまたは複数の断片を特徴付けて、共通バーコード配列の存在に少なくとも部分的に基づいて、前記1つまたは複数の断片を個々の細胞に帰属させることと、
(d)前記複数の離散区分における前記1つまたは複数の断片の前記特徴付けに基づいて、前記集団内の複数の個々の細胞を特徴付けることと、を含む、前記方法。 - 前記核酸を断片化することをさらに含む、請求項74に記載の方法。
- 前記離散区分が液滴である、請求項74に記載の方法。
- 前記核酸の前記1つまたは複数の断片を特徴付けることが、前記個々の細胞からのリボソームデオキシリボ核酸を配列決定することを含み、前記個々の細胞を特徴付けることが、細胞の属、種、株または変異体を識別することを含む、請求項74に記載の方法。
- 前記個々の細胞がマイクロバイオームサンプルに由来する、請求項77に記載の方法。
- 前記個々の細胞がヒト組織サンプルに由来する、請求項74に記載の方法。
- 前記個々の細胞が哺乳類の循環細胞に由来する、請求項74に記載の方法。
- 前記個々の細胞が法医学的サンプルに由来する、請求項74に記載の方法。
- 前記核酸が、前記離散区分の前記個々の細胞から放出される、請求項74に記載の方法。
- 個々の細胞または細胞集団を特徴付ける方法であって、
(a)複数の異なる細胞表面特徴結合基タイプであって、各異なる細胞表面結合基タイプは異なる細胞表面特徴に結合することができ、かつ存在する場合は、1つまたは複数の細胞表面特徴結合基とその細胞表面特徴それぞれとの間の結合を可能にする条件下で、それに結合したレポーターオリゴヌクレオチドを含む、前記複数の異なる細胞表面特徴結合基タイプを有する細胞をインキュベートすることと、 (b)前記細胞を、バーコード配列を含む複数のオリゴヌクレオチドを含む区分に区分することと、
(c)前記バーコード配列を前記区分に存在するオリゴヌクレオチドレポーター基に結合させることと、
(d)前記オリゴヌクレオチドレポーター基及び結合したバーコードを配列決定することと、
(e)配列決定されたレポーターオリゴヌクレオチドに基づいて前記細胞上に存在する細胞表面特徴を特徴付けることと、を含む方法。 - 複数の区分を含む組成物であって、前記複数の区分のそれぞれが、(i)個々の細胞、及び(ii)共通の核酸バーコード配列を含むオリゴヌクレオチドの集団を含む組成物。
- 前記複数の区分がエマルジョン中の液滴を含む、請求項84に記載の組成物。
- 前記複数の区分のそれぞれの中の前記オリゴヌクレオチドの集団が、前記複数の区分のそれぞれの中に配置されたビーズに結合される、請求項84に記載の組成物。
- 前記個々の細胞には、それらのそれぞれの細胞表面特徴に関連する複数の異なる細胞表面特徴結合基が結合しており、各異なるタイプの細胞表面特徴結合基は、異なるヌクレオチド配列を含むオリゴヌクレオチドレポーター基を含む、請求項84に記載の組成物。
- 前記複数の異なる細胞表面特徴結合基が、複数の異なる細胞表面特徴に対して結合親和性を有する複数の異なる抗体または抗体断片を含む、請求項87に記載の組成物。
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