GSM9553942: ChIPmentation of PBX1 on wt gastruloids at 120h; Mus musculus; ChIP-Seq
Collected gastruloid were pooled in a 15ml falcon tube, washed with PBS and resuspended in 1ml PBS containing 1% formaldehyde for fixation during 10 min at RT. The crosslink reaction was stopped by adding a glycine solution to a final concentration of 0.125M. Fixed gastruloids were pelleted and stored at -80°C until further use. Samples were resuspended in a sonication buffer (Tris HCl pH=8.0 50mM; EDTA 10mM; SDS 0.25% and protease inhibitors) and sonicated in a Covaris S220 device for 14 min (duty cycle 2%, peak incident power 105W) to obtain an average chromatin fragment size of 300-500 bp. A dilution buffer (HEPES pH=7.3 20mM; EDTA 1mM; NP40 0.1%; NaCl 150mM and protease inhibitors) was added to the sonicated chromatin and incubated with the antibody-bead complex (Pierce Protein A/G Magnetic Beads, Thermo Scientific with 5µg anti-PBX1 (4342S, Cell Signaling)) overnight at 4°C. Sequential washes were then performed twice with RIPA buffer (Tris HCl pH=8.0 10mM; EDTA 1mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 140mM and protease inhibitors), RIPA High salt buffer (Tris HCl pH=8.0 10mM; EDTA 1mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 500mM and protease inhibitors), LiCl buffer (Tris HCl pH=8.0 10mM; EDTA 1mM; LiCl 250mM; Sodium Deoxycholate 0.5%; NP40 0.5% and protease inhibitors) and Tris HCl buffer (pH=8.0 10mM and protease inhibitors). 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 2min with 1µl 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 on a DNBSEQ-G40 sequencer as paired-end, 100bp reads.