GSM7035384
Collected stembryos were pooled in a 15 ml falcon tube, washed with PBS and resuspended in 1 ml PBS containing 1% formaldehyde for fixation during 10 min at room temperature. The crosslink reaction was stopped by adding a glycine solution to a final concentration of 0.125 M. Fixed stembryos were pelleted and stored at -80°C until further use. ChIP and ChIP-M experiments were performed according to the protocol described in (Rodriguez-Carballo et al., 2017) and (Darbellay et al., 2019), and adapted for stembryos samples. Samples were resuspended in a sonication buffer (Tris HCl pH=8.0 50 mM; EDTA 10 mM; SDS 0.25% and protease inhibitors) and sonicated in a Covaris E220 device for 14 min (duty cycle 2%, peak incident power 105 W) to obtain an average chromatin fragment size of 300-500 bp. A dilution buffer (HEPES pH=7.3 20 mM; EDTA 1 mM; NP40 0.1%; NaCl 150 mM and protease inhibitors) was added to the sonicated chromatin and incubated with the antibody-bead complex (Pierce Protein A/G Magnetic Beads, Thermo Scientific) overnight at 4°C. Sequential washes were then performed twice with RIPA buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 140 mM and protease inhibitors), RIPA High salt buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 500 mM and protease inhibitors), LiCl buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; LiCl 250 mM; Sodium Deoxycholate 0.5%; NP40 0.5% and protease inhibitors) and Tris HCl buffer (pH=8.0 10mM and protease inhibitors). For ChIP experiments (H3K27ac (ab4729, Abcam), H3K27me3 (39155, Active Motif), CDX2 (sc-393572, Santa Cruz) and PolII-Ser2p (04-1571, Millipore)), DNA fragments were incubated in the elution buffer (Tris HCl pH=8.0 10 mM; EDTA 5 mM; NaCl 300 mM and SDS 0.1%) containing proteinase K and purified using the Qiagen MiniElute kit. A phosphatase inhibitor (PhosStop, Roche) was used during PolII-Ser2p ChIP incubation and beads wash. The DNA library was produced using TruSeq adapters and amplified with the Kapa HiFi library kit (Roche) using a number of cycles determined by qPCR. For ChIP-M samples (CTCF (61311, Active Motif), PolII (ab817, Abcam), RAD21 (ab992, Abcam) and NIPBL (A301-779A, Bethyl Laboratories)), DNA fragments bound to the antibody-bead complex were tegmented using the Nextera Tegmentation kit. Beads were resuspended in the tegmentation buffer and incubated at 37° for 2 min with 1 µl of the Tn5 transposase. Fragment were then eluted and purified as described previously, and amplified using Nextera primers. Final DNA libraries were purified and size selected using AMPure XP magnetic beads (Beckman Coulter), and a fragment analysis was performed before sequencing