Genome-wide Chromatin Profiling by ChIP-seq and ATAC-seq

Chromatin immunoprecipitation (ChIP) and Assay for Transposase-Accessible Chromatin (ATAC) sequencing was performed as previously described^{36 (link)}. For ChIP-seq a total of 10 × 10⁷ cells were crosslinked with 1% formaldehyde while shaking for 7 min at room temperature, quenched with 125 mM glycine, lysed and sonicated with the S2 Covaris for 30 min to obtain 200–300 bp long fragments. Chromatin fragments were immunoprecipitated overnight using 1 µg antibody of SOX11-PAb antibody (custom made by Absea biotechnology, China) and 20 µl Protein A UltraLink® Resin (Thermo Scientific, #53139) beads per 10 × 10⁷ cells. Reverse crosslinking was done at 65 °C for 15 h and chromatin was resuspended in TE-buffer, incubated for 2 h at 37 °C with 0.2 mg/ml RNase and followed by an incubation of 2 h at 55 °C with 0.2 mg/ml proteinase K. DNA was isolated using 400 µl phenol:chloroform:isoamylalcohol (P:C:IA) in phase lock gel tubes (5Prime). Upon centrifugation, the aqueous layer was transferred to a new tube with 200 mM NaCl, 30 µg glycogen and 800 µl 100% ethanol, and incubated for 30 min at −20 °C. Upon centrifugation, the pellet was washed with 80% Ethanol and resuspended in RNase/DNase free water. DNA concentration was measured using the Qubit® dsDNA HS Assay Kit. Library prep was done using the NEBNExt Ultra DNA library Prep Kit for Illumina (E7370S) with 500 ng starting material and using 8 PCR cycles according to the manufacturer’s instructions. For ATAC-seq, 50,000 cells were lysed and fragmented using digitonin and Tn5 transposase. The transposed DNA fragments were amplified and purified using Agencourt AMPure XP beads (Beckman Coulter). ChIP and ATAC library concentrations were measured with the Illumina Kapa Library quantification kit (Roche #07960140001) and libraries were sequenced on the NextSeq 500 (Illumina) using the Nextseq 500 High Output kit V2 75 cycles single-end (Illumina).