title: GSM5627334: Pcgf2fl_ES_UNT_rep3_CapC_set1; Mus musculus; OTHER
library_construction_protocol: CaptureC libraries were prepared as described previously (Hughes et al., Nature Genetics 2014). Briefly, 10 Million mouse ES cells were trypsinized, collected in 50ml falcon tubes in 9.3ml media and crosslinked with 1.25ml 16% formaldehyde for 10min at room temperature. Cells were quenched with 1.5M glycine, washed with PBS and lysed for 20 minutes at 4oC lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40, supplemented with complete proteinase inhibitors) prior to snap freezing in 1ml lysis buffer at -80oC. Lysates were then thawed on ice, pelleted and resuspended in 650µl 1x DpnII buffer (NEB). Three 1.5ml tubes with 200µl lysate each were treated in parallel with SDS (0.28% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off), quenched with trypsin (1.67% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off) and subjected to a 24 hour digestion with 3x10µl DpnII (homemade, 37 oC, interval shaking 500rpm, 30sec on/30sec off). Each chromatin aliquot was independently ligated with 8 µl T4 Ligase (240 U) in a volume of 1440µl (20 hours, 16oC). Following this, the nuclei containing ligated chromatin were pelleted to remove any non-nuclear chromatin, reverse-crosslinked and the ligated DNA was phenol-chloroform purified. The sample was resupended in 300µl water and sonicated 13x (Pico Bioruptor, 30sec on, 30sec off) or until a fragment size of approximately 200bp was reached. Fragments were size selected using AmpureX beads (Beckman Coulter: A63881, selection ratios: 0.85x / 0.4x). 2x 1-5µg of DNA were adaptor ligated and indexed using the NEBNext DNA library Prep Reagent Set (New England Biolabs: E6040S/L) and NEBNext Multiplex Oligos for Illumina Primer sets 1 (New England: E7335S/L) and 2 (New England: E7500S/L). The libraries were amplified 7x using Herculase II Fusion Polymerase kit (Agilent: 600677). Libraries were hybridized in teh following way: Probes were designed using the online tool by the Hughes lab (CapSequm: http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi) to be either 120bp (Sox2) or 70bp long each, two probes for each promoter of interest. The probes were pooled at a 2.9nM each and the samples were multiplexed by mass prior to hybridization. Hybridization was carried out using the Nimblegen SeqCap system (Roche, Nimblegen SeqCap EZ HE-oligo kit A # 06777287001, Nimblegen SeqCap EZ HE-oligo kit B # 06777317001, Nimblegen SeqCap EZ Accessory kit v2 # 07145594001, Nimblegen SeqCap EZ Hybridisation and wash kit # 05634261001) according to Roche protocol for 72 hours followed by a 24 hours hybridization (double Capture). The captured libraries were sequenced on Illumina NextSeq. CaptureC