title: GSM2974348: K27-DMOG-d9; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-sequencing was performed as described previously with modifications (Sridharan et al., 2013). Briefly, NPCs were crosslinked by 1% formaldehyde (final concentration) for 10 minutes at room temperature, and quenched with 125mM glycine (final concentration). Cells were re-suspend in sonication buffer containing 50 mM Hepes-KOH (pH=7.9), 140 mM NaCl, 1mM EDTA (pH=8), 1% Triton X-100, 0.1% sodium Deoxycholate, 1% SDS and protease inhibitor, and sonicated with Misonix sonicator S-4000. Cell extracts were incubated with 5µg antibodies against H3K27Ac (Abcam, ab4729) or H3K9Me3 (Abcam, ab8898) overnight at 4 °C. Immunoprecipitates were collected with magnetic beads, and washed twice with PBS, low-salt buffer, high-salt buffer and LiCl buffer as described before (Sridharan et al., 2013). Reverse crosslinking was preformed by addition of 1% SDS and proteinase K with gentle shaking overnight at 56°C. DNA was precipitated using phenol-chloroform-isoamyl alcohol mixture (Sigma, P2069) and Phase Lock Gel (5 Prime) according to manufacturer's protocol. ChIP-seq library preparation was performed using Ovation Ultralow System V2 1-16 (NuGEN) following the user's guide. The sequencing was performed on HiSeq 2000 platform with single-end 50bp run.
attributes.tag: GEO Accession
attributes.value: GSM2974348