GSM9241253: ChIP-seq, iPS cells, H3K27ac, RETT, s3042, Rep1; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed according to a protocol published by Agilent technologies (Santa Clara, CA, http://www.agilent.com). Briefly, iPS cells were fixed by 1% formaldehyde solution for 10 min. After harvesting the fixed cells, the cells were lysed in lysis buffer, and sonicated using Misonix XL2020 sonicator (MISONIX Inc., NY, http://misonix.com) until the DNA fragments were 200–600 base pairs in length. 3.0% of the total volume was stored as input at −20°C, until use. Immunoprecipitation was performed at 4°C, overnight with antibodies described bellow. DNA/beads was washed with a low salt buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100) once, and then further washed with a high-salt buffer (20 mM Tris-HCl pH 7.4, 400 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) once before washing with a RIPA buffer. Immune complexes were disrupted with direct elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) and the covalent links between immunoprecipitates and input chromatin were disrupted by incubation at 65°C for overnight. DNA was further incubated with RNaseA and proteinase K (Nacalai Tesque), purified by phenol extraction, and ethanol precipitated. DNA pellets were dissolved in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA pH8.0). The antibodies were used anti-CTCF(3418S, Cell Signaling Technology) and Acetyl-Histone H3 (Lys27) (H3K27ac, 8173S, Cell Signaling Technology) A sequencing library was prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB) according to the manufacturer's instructions.