US 12,377,396 B2
Systems and methods for multiplexed measurements in single and ensemble cells
Naresha Saligrama, Stanford, CA (US); Ansuman Satpathy, Stanford, CA (US); Howard Y. Chang, Stanford, CA (US); and Mark M. Davis, Stanford, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Appl. No. 16/967,402
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
PCT Filed Dec. 19, 2018, PCT No. PCT/US2018/066592
§ 371(c)(1), (2) Date Aug. 4, 2020,
PCT Pub. No. WO2019/152108, PCT Pub. Date Aug. 8, 2019.
Claims priority of provisional application 62/626,603, filed on Feb. 5, 2018.
Prior Publication US 2021/0213413 A1, Jul. 15, 2021
Int. Cl. B01J 19/00 (2006.01); C12N 15/10 (2006.01); C12Q 1/6874 (2018.01)
CPC B01J 19/0046 (2013.01) [C12N 15/1096 (2013.01); C12Q 1/6874 (2013.01); B01J 2219/00596 (2013.01); B01J 2219/00722 (2013.01); C12Q 2537/165 (2013.01)] 28 Claims
 
1. A method of processing immune cells, comprising:
(a) isolating a single immune cell comprising genomic deoxyribonucleic acid (gDNA) and a plurality of messenger ribonucleic acid (mRNA) molecules;
(b) tagmenting one or more regions of accessible chromatin in the gDNA of the single immune cell to produce a plurality of tagged gDNA fragments in a tagmentation reaction, wherein the tagmentation reaction comprises cleaving and tagging the gDNA in the same reaction;
(c) adding a primer, reverse transcriptase, and dNTPs to the single immune cell to reverse transcribe the plurality of mRNA molecules of the single immune cell to produce a plurality of complementary DNA (cDNA) molecules, wherein said cDNA molecules comprise sequences that correspond to a V(D)J region of a genome of the single immune cell;
(d) sequencing the plurality of cDNA molecules and the plurality of tagged gDNA fragments that were both produced from the single immune cell to generate a plurality of sequencing reads; and
(e) generating an epigenetic map of the one or more regions of accessible chromatin of the gDNA and the sequences that correspond to the V(D)J region of the single immune cell by correlating:
one or more sequencing reads of the plurality of tagged gDNA fragments or amplicons thereof; and
one or more sequencing reads of the plurality of cDNA molecules or amplicons thereof.