GSM3819697: TxCL63_Dox24h_rep2_CutRun_Spen; Mus musculus; OTHER
10 million cells in suspension were fixed with 2% formaldehyde diluted in PBS for 10 minutes at room temperature. Fixation was quenched with 125mM glycine for 5 minutes and cells were washed twice with PBS. Fixed cells were then permeabilized with permeabilization buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 0.25% TritonX-100, c0mplete EDTA free) for 5 minutes and washed twice in PBS. Cells were then resuspended in washing buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, cOmplete EDTA free) and bound to concanavalin beads (50uL bead slurry used per 10 million cells), blocked in blocking buffer (wash buffer + 2mM EDTA) and incubated overnight with target antibodies diluted 1/200 in washing buffer on an end to end rotator. Cells were washed three times in washing buffer followed by 1-hour incubation with pA-MNase (1:400 in washing buffer) and washed again three times in washing buffer. After the last wash, cells were resuspended in washing buffer and left equilibrated to 0°C in a metal block for 10 minutes. To start digestion, CaCl2 was added to 1.5mM final concentration, taking care to return each sample to 0°C immediately after. Digestion was performed at 0°C for 1 hour, before being stopped by adding 2X-STOP solution (200mM NaCl, 20mM EDTA, 5mM EGTA, 0.1% NP-40, 40ug/mL glycogen). RNAse A was added to a final concentration of 50ug/mL and samples were incubated at 37°C for 20 minutes. SDS and proteinase K were then added to final concentrations of 0.1% and 300ug/mL respectively and samples were incubated at 56°C for 2 hours followed with 68°C for 16 hours to reverse crosslinking. Total DNA was extracted using phenol-chloroform followed by several rounds of ethanol precipitation and DNA size selection (using Ampure XP beads) to remove predominating large undigested DNA fragments. The selected small fragments (resulting from MNase digestion) were quantified and analyzed using Qubit and Tapestation assays. Cut&Run libraries were prepared from 50ng DNA per samples, using the Accel-NGS 2S Plus DNA Library Kit (Swift) according to manufacturer's protocol. Paired-end 100nt sequencing was performed on HiSeq2500 (Illumina, San Diego, CA).