GSM5065150: Pcgf1_f/f_LSK_MNaseSeq_1; Mus musculus; MNase-Seq
MNase-seq:One million cells were cross-linked by adding formaldehyde to 1% and incubated for 10 min at RT, quenched using 1 M glycine, and washed three times in PBS. Collected cells were lysed in 1 ml PBS + 0.5% Triton X-100 for 3 min on ice. Nuclei were pelleted and washed by MNase digestion buffer (10 mM Tris-HCl, pH 7.4, 15 mM NaCl, 60 mM KCl, 0.5 mM spermidine), spun down and resuspended in the same buffer. Ca2+ concentration was adjusted to 1mM with 1M CaCl2 and MNase was added to 10 U/ml. The reaction mixture was incubated at 37°C for 10 min and the reaction was stopped by adding 150 μL of stop buffer (20 mM EDTA, 20 mM EGTA, 0.4% SDS, 0.5 mg/ml proteinase K) and then incubated for overnight at 65°C. DNA was purified using a MinElute PCR purification Kit (28006; QIAGEN) and loaded to 1.5% Agarose Gel. Mononucleosomal bands were excised and purified by a gel extraction kit (28705; QIAGEN). Fragmented DNAs fragmentd by MNase were subjected to library preparation by NEBNext UltraⅡ DNA library preparation kit for Illumina(E7645; New England Biolabs). Preparation of scRNA-seq library: FACS sorted LSKs (control and Pcgf1-KO) were subjected to preparation of individually barcoded single-cell RNA-Seq libraries using the Chromium instrument and the Single Cell 3′ Reagent kit following the manufacturer's instructions (10X Genomics).