GSM2101764: 3691_RBPJ; Homo sapiens; ChIP-Seq
For histone modification and transcription factor ChIP-Seq, 2 million cells (H3K27Ac) or 10 million cells (RBPJ) were crosslinked in PBS + 1% fresh formaldehyde for 10 minutes at 37 C, quenched for 5 minutes with 125 mM glycine, washed twice in cold PBS with protease inhibitors (complete PI, Roche), and stored at -80 C. Pellets were thawed 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 , resuspended in cold SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 + PI) for 10 minutes, sonicated to an average fragment size of 200-400 bp on a Branson sonifier, and cleared of debris by centrifugation. Samples were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 0.25% 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 towards H3K27ac (Abcam, ab4729) or RBPJ (Abcam, no. 25949 ). Antigen-antibody complexes were collected with protein G Dynabeads (Life technologies) for 4 hours at 4 C, and sequentially washed with RIPA buffer (0.1% Na deoxycholate, 0.1% SDS, 1% Triton x-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), RIPA/High salt (0.1% Na deoxycholate, 0.1% SDS, 1% Triton x-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 mM NaCl), LiCl Wash Buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE Buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Beads were resuspended in Low SDS ChIP elution buffer (10mM Tris-HCl pH 8.0, 0.5M EDTA, 300mM NaCl, 0.1% SDS, 5mM DTT) and incubated for 6 hours at 65 C to elute DNA and reverse crosslinking. Samples were treated with RNAase for 30 minutes and proteinase K for 2 hours at 37 C. ChIP DNA was then purified from supernatants with AMPure beads (Beckman-Coulter). Input DNA was prepared in parallel by adding unenriched, diluted chromatin directly into the elution / reverse crosslinking step. ChIP DNA was end-repaired (End-It, Epicentre), A-tailed (Klenow fragment 3'-->5' exo-, New England Biolabs), and ligated to barcoded illumina adaptors (Quick T4 DNA ligase, NEB). Each reaction was followed by clean-up with AMPure beads (Beckman-Coulter). Ligation products were amplified by PCR for 14-18 cycles with illumina primers and PFU Ultra II HS PCR mix (Agilent). Library size selection to 300-600 bp was performed by two-step AMPure bead selection or gel purification (E-Gel SizeSelect 2%, Life technologies).
GEO
GSM2101764