GSM6398672: UoCB6_ETV6_Atlas_ChIP-Seq; Homo sapiens; ChIP-Seq
Twenty million cells were cross-linked in PBS + 1% formaldehyde for 10 minutes at 37C (TFs), quenched with 1/20th volume of 2.5M glycine, washed twice in cold PBS with protease inhibitors, and lysed in cold cytoplasmic lysis buffer (20 mM Tris-HCl ph8.0, 85 mM KCl, 0.5% NP 40 + PI). Nuclei were pelleted at 3000g, and resuspended in cold SDS lysis buffer (0.3% SDS for H3K27ac and 1% SDS for TFs, 10mM EDTA, 50mM Tris-HCl, pH 8.1 + PI) for 10 minutes. Nuclei were fragmented on a Q800R2 Sonifier (QSonica) as follows: three cycles of amplitude = 50, pulse times: 30s on / 30s off, total on time = 3:20 m, temperature = 80C (histone marks) or two cycles of amplitude = 70, pulse times: 45s on / 15s off, total on time = 8:50 m, temperature = 40C (TFs). Samples were diluted 1:3 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl +PI), and rotated at 4°C overnight with 2-5 ug of antibody. Antibody-chromatin complexes were captured with protein G–conjugated beads, washed, and eluted. After reversal of cross-linking, RNase and proteinase K treatment were performed and DNA was purified with Ampure XP beads for library preparation. Input sample was prepared by the same approach without immunoprecipitation. ChIP DNA was end-repaired (End-It, Epicentre), A-tailed (Klenow fragment 3'-->5' exo-, New England Biolabs), and ligated (Quick T4 DNA ligase, NEB) to barcoded illumina adaptors (Single-Indexed Adapter Kit, KAPA, #KK8700). Each reaction was followed by clean-up with AMPure XP beads. Ligation products were amplified by PCR for 14-18 cycles with illumina indexing primers and PFU Ultra II HS PCR mix (Agilent). Library size selection to 300-600 bp was performed by two-step AMPure bead selection.