GSM4066635: NHDF_ChIP-seq; Homo sapiens; ChIP-Seq
Cells were scraped off in PBS containing 0.5% BSA and collected into a 50-mL Eppendorf tube. Pellet cells at 2,000 rpm for 8 min, and transfer cells into 1.5 mL tubes. Cells were lysed in sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, cocktail) and sonicated using Bioruptor (Diagenode). The supernatant of Lysate was diluted at 1:10 using RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-doxycholate, cocktail), and incubated with Dynabeads M-280 streptavidin (11206D, Thermo Fisher Scientific) for affinity purification. The beads were washed once with low salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), once with high salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), once with LiCl buffer (250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were collected into new EP tubes with TE buffer and incubated on a Mixing Block at 65 ℃ overnight at 1,000 rpm to reverse the formaldehyde cross-links. The supernatant was incubated with 200 ng/μL of RNase A at 37 ℃ for 1 h and then 200 ng/μL of proteinase K at 55 ℃ for 2 h. DNA was extracted with phenol and precipitated with ethanol using 30 μg glycogen (10901393001, Roche) as the carrier. DNA pellet was dissolved in TE buffer Libraries were prepared according to Illumina's instructions accompanying the NEBNext® Ultra™ DNA Library Prep Kit. Libraries were sequenced on an Illumina HiSeq™ 2500 platform with 6G base PE150 reads.
GEO Accession
GSM4066635