alias: GSM3595838
title: GSM3595838: ChIP-seq-NALM6-a-H3K27ac-1; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-Seq:  10M cells for transcriptions factor ChIP were fixed at RT in 1 mg/ml DSG (ThermoFisher Scientific) in PBS for 30 min followed by an additional 10 min after addition of formaldehyde up to 1%. Cells (10M) for Histone modification ChIP were fixed in 1% formaldehyde for 10 min. The reactions were quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed in PBS. Nuclei were isolated by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5% NP40) + protease inhibitor cocktail (PIC) (1X Roche protease inhibitors – 11697498001) on ice. Pelleted nuclei were dissolved in Lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8)) + PIC and sonicated on a Bioruptor (Diagenode). Sonication was followed by pelleting of debris and the supernatant was transferred to new tube and chromatin was diluted 5X in Dilution Buffer (1% Triton, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8) + PIC) for transcription factor ChIP and in HBSS (Lonza, Verviersa, Belgium) +PIC and 2X RIPA buffer (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% Sodiumdeoxycholate, 200 mM NaCl) + PIC for histone modification ChIPs. Ten μg per 10M cells of antibody Rabbit anti-Ikzf1 polyclonal IgG [ab26083, Abcam], Rabbit polyclonal anti-Ebf1 [ABE1294, Millipore], Rabbit polyclonal anti-Pax5 [ab183575, Abcam], Rabbit polyclonal anti-RUNX1 [ab23980, Abcam] or 10μl of H3K4Me3 polyclonal IgG [07-473 Millipore], or 10μg of Rabbit anti H3K27Ac IgG [ab4729, Abcam] was hybridized to 70μl Protein-G or A Dynabeads (Life Technologies). ChIP was performed over night at 4°C, and subsequently washed (1 time with 500 μl Low Salt Immune Complex Wash Buffer, 1 time with 200 μl High Salt Immune Complex Wash Buffer, 1 time with 200 μl LiCl Immune Complex Wash Buffer, 2 times with 200 μl TE buffer) and eluted for 6 h at 65°C (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, 100 μg RNase A and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator before ChIP-qPCR or ChIP-seq library preparation using NEXTflex DNA barcodes (BIOO scientific). 76 bp single read sequencing was performed on an Illumina NextSeq500.
identifiers.namespace: GEO
identifiers.id: GSM3595838