GSM7729029: Mouse Female Day 4 EpiLC R1 – XIST Pulldown; Mus musculus; OTHER
5× 106cells were harvested per condition after accutase-mediated dissociation and RAP-seq was performed, based on the protocol adapted from Engreitz et al. (Engreitz et al., 2013). Briefly, cells were incubated for 45min with freshly made 2mM DSG in PBS at room temperature, crosslinked with 3% formaldehyde for 10min and quenched with 500mM glycine. Cells were subsequently pelleted at 1,500xg for 5min at 4˚C, aliquoted into vials of 10 million cells each, and subjected to lysate preparation as follows: Cell pellets were resuspended in 10ml of nuclear extraction buffer LB1 containing50mM HEPES-KOH (pH 7.5), 140mM NaCl, 1mM EDTA, 10% (vol/vol) glycerol, 0.5% (vol/vol) NP-40/Igepal CA-630 and 0.25% (vol/vol) Triton X-100, and incubated for 10min with rotation at 4°C, then pelleted at 2000xg for 5min and the same procedure was performed with buffer LB2, which contains 10mM Tris-HCL (pH 8.0), 200mM NaCl, 1mM EDTA and 0.5mM EGTA. For cell lysis, nuclei were resuspended in 300μL of buffer LB3 containing 10mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% (wt/vol) sodium deoxycholate and 0.5% (vol/vol) N-lauroylsarcosine, then sonicated on ice using the Misonix S-400 sonicator with microtip (model number: U1240A0418) for 2min at an amplitude of 12 with 1sec pulses intermitted by 3sec pauses. Next, chromatin was further digested using TURBO DNase at a concentration of 0.1-0.4U/μl at 37°C for 15min. The digestion was stopped by the addition of DNase Stop Solution (mixture of EDTA and EGTA). 5% of each sample was set aside as the input control, while the remaining sample was used for the actual pulldown. The RNA pulldown was performed using 50pmol of 90nt-long biotinylated oligonucleotide probes for every 5× 106cells and Streptavidin C1 beads. DNA was eluted by RNase H digestion, and the crosslinks were reversed by proteinase K digestion of the eluted DNA at 60°C. DNA libraries were prepared using NEB Next Ultra End Repair/dA-Tailing Module (NEB) and TruSeq DNA adapters (Illumina) were ligated using Quick Ligase (NEB). Libraries were amplified by KAPA HiFi Polymerase, pooled and sequenced on the Illumina HiSeq 6000 platform to generate 50bp pair-end reads.