Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Primary hematopoietic stem/progenitor cells were isolated form bone marrow and Lineage-negative/c-Kit-positive (HSPCs) were enriched by MACS. Nuclei were isolated, washed and frozen in wash buffer according to the “Bench top CUT&Tag V.3” protocol (https://ww.protocols.io/view/bench-top-cut-amp-tag-bcuhiwt6). For the actual CUT&Tag experiment, we followed the CUT&Tag-direct protocol described by Henikoff et al.74 using the CUTANA pAG-Tn5 enzyme (Epicypher). Briefly, aliquots of 30,000-45,000 native nuclei per reaction were bound to activated Concanavalin A beads. After successive incubations with primary antibody (overnight at 4°C) and secondary antibody (0.5-1 h) in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1x protease inhibitor), the beads were washed and resuspended in pAG-Tn5 (1:20 dilution) in 300-wash buffer (wash buffer containing 300 mM NaCl) for 1 h. Incubations were performed at room temperature, except when otherwise stated, in volumes of 25-50 ul in low-retention PCR tubes. Tagmentation was performed for 1 h in 300-wash buffer supplemented with 10 mM MgCl2. Following tagmentation, beads were washed in 50 ul TAPS buffer (10 mM TAPS pH8.5, 0.2 mM EDTA), resuspended in 5 ul SDS release buffer (0.1% SDS, 10 mM TAPS pH8.5) and incubated for 1 h at 58°C. SDS was neutralized with 15 ul of 0.67% Triton-X100, and 4 ul of dual indexed primers from the “IDT for Illumina Nextera DNA UD Indexes Set A” (Illumina) as well as 25 ul of NEBNext High-Fidelity 2x PCR Master mix (NEB) were added. Gap filling and 18 cycles of PCR were performed, followed by clean-up with 65 ul of SPRIselect beads (Beckman Coulter). CUT&Tag