GSM7665008: HSC_old_CD150_low_H3K27ac_rep1; Mus musculus; OTHER
The sorted HSCs (2,000-5,000) were immediately used for CUT&RUN assay. The H3K4me3 (Cell Signaling Technology, # 9751S) and H3K27ac (EMD Millipore #07-360) CUT&RUN libraries were prepared. Briefly, the FACS-sorted HSCs were collected in a 1.5-ml tube containing 50 μl of wash buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitor cocktails (Sigma-Aldrich)]. The HSCs were then captured with BioMagPlus Concanavalin A (Polysciences) beads and incubated with a primary antibody overnight at 4°C in antibody incubation buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktails (EDTA-free tablet, Roche), 2 mM EDTA, and 0.005% digitonin (Life Technologies)]. The H3K4me3 antibody (1:100) and H3K27ac antibody (1:100) were used. After unbound antibodies were washed away, protein A–MNase was added at a 1:200 ratio (500 ng/ml) and incubated for 3 hours at 4°C. After washing, CaCl2 was added to a final concentration of 2 mM to activate pA-MN, incubated for 20 min at 4°C, and then stopped by adding 1/10 volume of 10× STOP buffer [1.7 M NaCl, 100 mM EDTA, 20 mM EGTA, RNase A (250 μg/ml; Invitrogen), and glycogen (250 μg/ml; Invitrogen)]. The protein-DNA complexes were released by incubating at 37°C for 10 min, followed by 16,000g centrifugation for 5 min at 4°C. The supernatant was transferred to a new Lo-bound tube, 1/100 volume of 10% SDS, and 1/80 volume of Proteinase K (25 mg/ml; Life technologies) were added and incubated at 55°C for at least 1 hour. DNA was then precipitated by phenol/chloroform/isoamylalcohol (25:24:1), followed by ethanol precipitation with glycogen, and then dissolved in water. Sequencing libraries were then prepared using the NEBNext Ultra II DNA library preparation kit for Illumina (New Engliand Biolabs) according to the manufacturer's instructions. The libraries were sequenced on the Illumina NextSeq 550 with pair-end 75-bp reads.