GSM8084967: GB-Hybrid_H3K27me3; Homo sapiens; ChIP-Seq
Full single cell ChIP-seq protocol has been previously described in (Grosselin, 2019). Briefly, 1M cells were labeled by 20 min incubation of 1 μM CellTrace solution (CellTrace CFSE, Thermo Scientific, C34554) at room temperature. Cells were then washed and incubated in culture medium for 15 minutes to allow the CellTrace reagent to undergo acetate hydrolysis. Labeled cells were resuspended in a suspension mix (PBS supplemented with 30% Percoll, 0.1% Pluronic F-68, 25 mM HEPES pH 7.4 and 50 mM NaCl) and encapsulated in 45pL droplets with a digestion mix (0,1 M Tris-HCl pH 7.4, 0,3 M NaCl, 2,15% Tritonx100, 0,22% NaDoc, 0,01 M CaCl2, 0,2 U/µl Micrococcale Nuclease (Thermo Scientific, EN0181), 1x Protease Inhibitor Cocktail and 2µM Sulforhodamine B). Once encapsulated, cells droplets are collected and incubated at 37°C for 20 minutes for cellular lysis and chromatin digestion. In parallel, beads with unique double-stranded barcodes were encapsulated in 100pL droplets with a ligation mix (0,1 M EGTA, 2 mM ATP, 2 μM Sulforhodamine B, 0,38 U/μL Fast link DNA ligase (Lucigen, LK0750H)) and a repair mix (4 mM dNTP, 2 μM Sulforhodamine B, 0,1 x End-it Repair mix (Lucigen, ER81050), 0,1 U/μL Fast link DNA ligase (Lucigen, LK0750H)). Beads droplets and cells droplets were then fused with a 1:1 ratio by electrocoalescence and counted by laser detection as previously described (Grosselin, 2019).  Double-stranded DNA barcodes were released in fused droplets by UV exposure (1 min, 230mW/cm2) and ligated to nucleosomes during an overnight incubation at 16°C. Immunoprecipitation of barcoded nucleosomes associated with H3K27me3 histone mark (Antibody Cell Signaling Technology, 9733-C36B11, 1/200) or H3K4me3 histone mark (Antibody Cell Signaling Technology, 9751-C42D8, 1/200) was performed overnight at 4°C on a rotating wheel. DNA amplification and library preparation were performed as described in (Grosselin, 2019). Libraries were then sequenced on the NovaSeq 6000 in PE100, with a coverage of 100,000 reads per cell.