NextSeq 500 paired end sequencing; Capture-C experiments on auxin treated control (E14-Tir) and Scc1-AID, Ring1b-AID, Ring1b-AID-Scc1-AID  and CTCF-AID (with and without a 6-hour auxin treatment) cells
Capture-C experiments on auxin treated control (E14-Tir) and Scc1-AID, Ring1b-AID, Ring1b-AID-Scc1-AID  and CTCF-AID (with and without a 6-hour auxin treatment) cells
2. Briefly, 107 mouse ES cells were trypsinized, collected in 50ml falcon tubes in 9.3ml media and crosslinked with 1.25ml 16% formaldehyde (Thermo Scientific, Cat. No 10751395) for 10min at room temperature. Cells were quenched with 1.5ml 1M glycine, washed with PBS and lysed for 20 minutes at 4oC while rotating (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 (homemaid, 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 and reverse-crosslinked. 1. Wild type E14 cells were grown on gelatin-coated plates in DMEM (Gibco, Carlsbad, CA) supplemented with 10% FBS, 10 ng/mL leukemia-inhibitory factor, penicillin/streptomycin, beta-mercaptoethanol, L-glutamine and non-essential amino-acids. Cells were treated with Nocodazole (100ng/ml) for 6 hours. 3. Ligated DNA was phenol-chloroform purified. 4. 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) and the correct size was assessed by Bioanalyzer. 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).
Experimental Factor: genotype
E14_Tir1