GSM5211730: Αlpha6+/Sca1-_H3K27me3; Mus musculus; ChIP-Seq
Αlpha6+/Sca1- cells were briefly homogenized in DMEM media with 10% FBS serum/1M sodium butyrate and cross-linked with 1% formaldehyde at room temperature with rotation for 15 min. Cross-linking was quenched with 0.125 M glycine, and HFSC were rinsed with cold 1 X PBS, re-suspended in 500 µl of sonication buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycho-late, and 0.5% N-lauroylsarcosine) with 1% SDS, and incubated at 4C for 10 min. Chromatin was sonicated to generate DNA fragments of 200–500 bp. Sonicated chromatin was incubated overnight with 5 μg of antibodies against H3K27me3 (39536; Active Motif) or H3K4me3 (39159; Active Motif; Rada-Iglesias et al., 2011; Rehimi et al., 2016), followed by 4–6 hr incubation with Protein G Dynabeads (Life Technologies). Beads were washed four times with 1 mL of cold radioimmunoprecipitation assay buffer (RIPA) washing buffer (50 mM HEPES, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Na-deoxycholate) and once with Tris/EDTA buffer (TE) containing 50mM NaCl. Immunoprecipitated chromatin was eluted from the beads by adding elution buffer (50 mM Tris, 10 mM EDTA, and 1% SDS) and vortexing at 65C for 15 min. Crosslinking was reversed by incubating the eluted samples at 65C overnight. Samples were then diluted (1:1) with TE and sequentially treated with RNase A (0.2mg/mL) and proteinase K (0.2mg/mL). DNA was purified using phenol:chloroform, followed by ethanol precipitation, and re-suspended in water. DNA libraries from H3K27me3 ChIP, H3K4me3 ChIP, and corresponding input DNA samples were generated with the TruSeq kit (Illumina) as single reads