GSM8819453: Vps15, mouse embryonic fibroblasts, 4h methionine starvation, rep2; Mus musculus; OTHER
Cells were scraped from plates in Cut and Run Wash Buffer with protease and phosphotase inhibitors and briefly fixed with 0.1% formaldehyde in DPBS for 3min. After washing with Wash Buffer, cells were attached to Concavalin A beads, and then incubated with respective antibodies for 1h. Cells attached to beads were then washed twice with Wash Buffer and then incubated with pAG-MNase for 10min, and washed twice again prior to putting on ice and adding Ca2+ to each well to inititate the cutting of pAG-MNase. Ca2+ was quenched with Stop  Buffer and DNA fragments eluted at 37C. DNA was purified with phenol-chloroform extraction prior to library preparation, quality control, and sequencing. DNA fragments were subject to end-repair and 3' adenylation. PCR products were enriched using Agencourt AMPure XP-medium kit. dsDNA PCR products were denatured and circularized by the splint oligonucleotide sequence. Single-stranded circular DNA was formatted as the final library, and DNA nanoballs were made from the library and loaded into a patterned nanoarray. Paired-end reads were generated by sequenced combinatorial Probe-Anchor-Synthesis with the DNBSEQ-400 sequencer.