ChIP-sequencing (ChIP-seq)

HSCs purified as indicated above were sorted and crosslinked with 1% formaldehyde at room temperature (RT) for 10 min, and the reaction was stopped by 0.125M glycine at RT for 5 min. Cross-linked cells were lysed and sonicated to 200–500 bp fragments (Bioruptor, Diagenode). ChIP-qualified antibodies (H3K4me3 Millipore 07-473, H3K27me3 Millipore 07-449) were added to the sonicated chromatin and incubated at 4°C overnight. Following this, 10 μl of protein A magnetic beads (Dynal, Invitrogen) previously washed in RIPA buffer were added and incubated for an additional 2 hours at 4°C. The bead: protein complexes were washed three times with RIPA buffer and twice with TE buffer. Following transfer into new 1.5 ml collection tube, genomic DNA was eluted for 2 hours at 68°C in 100 μl Complete Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 μg/ml proteinase K), and combined with a second elution of 100 μl Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl) for 10 min at 68°C. ChIPed DNA was purified by MinElute Purification Kit (Qiagen) and eluted in 12 μl elution buffer. ChIPed DNA was successfully made into a library using ThruPLEX-FD preparation kit (Rubicon, Ann Arbor, MI). Sequencing was performed according to the manufacturer’s protocol on a HiSeq 2000 (Illumina). Sequenced reads were mapped to the mm9 mouse genome and peaks were identified by model-based analysis of ChIP-seq data (MACS).⁶⁰