US 12,275,936 B2
Method to connect chromatin accessibility and transcriptome
Kun Zhang, La Jolla, CA (US); and Song Chen, La Jolla, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Appl. No. 17/044,128
Filed by The Regents of the University of California, Oakland, CA (US)
PCT Filed Apr. 18, 2019, PCT No. PCT/US2019/028042
§ 371(c)(1), (2) Date Sep. 30, 2020,
PCT Pub. No. WO2019/204560, PCT Pub. Date Oct. 24, 2019.
Claims priority of provisional application 62/659,210, filed on Apr. 18, 2018.
Prior Publication US 2021/0123044 A1, Apr. 29, 2021
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6876 (2018.01)
CPC C12N 15/1065 (2013.01) [C12Q 1/6876 (2013.01); C12Q 2600/158 (2013.01)] 8 Claims
OG exemplary drawing
 
1. A method for concurrent characterization of gene expression levels and epigenetic landscape within a single cell by determining chromatin accessibility and RNA expression in the cell, the method comprising:
a) labeling chromatin open regions in the cell's intact extracted nucleus using Tn5 transposase to add an adapter sequence to the chromatin open regions;
b) combining a splint oligonucleotide and barcoded oligo-dT beads comprising strings of deoxythymine in lysis buffer, wherein the splint oligonucleotide comprises a 5′ region complementary to the adapter sequence and a 3′ poly (A) tail;
c) co-encapsulating the nucleus and barcoded oligo-dT beads in the lysis buffer to form a plurality of droplets;
d) releasing the Tn5 transposase from genomic DNA within the droplets;
e) retrieving barcoded beads from droplets after releasing the Tn5 transposase, wherein the retrieved barcoded beads comprise 1) mRNA from the cell annealed to the strings of deoxythymine and 2) the genomic DNA from the cell annealed via the adapter sequence to the splint oligonucleotide, wherein the splint oligonucleotide is further annealed to the strings of deoxythymine via the 3′ poly (A) tail;
f) subjecting the retrieved barcoded beads to gap filling/ligation of chromatin and reverse transcription of mRNA; and
g) preparing single nucleus chromatin and RNA sequencing libraries by detecting the barcoded beads.