alias: GSM3377376
title: GSM3377376: Cochlea_IgG_control; Mus musculus; ChIP-Seq
library_construction_protocol: Cochleae were dissected from E13.5-14.0 embryos and cross-linked with 1% formaldehyde at room temperature for 30 mins.  Samples were then homogenized and lysed in cold lysis buffer  (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors) and gently rocked at 4°C for 10 minutes in 15 ml conical tubes. Cells were pelleted at 2000 g at 4°C and resuspended in cold wash buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors) and gently rocked at 4°C for 10 minutes in 15 ml conical tubes. Cells were pelleted at 2000 g at 4°C in a table top centrifuge and resuspended in 1 ml cold sonication buffer (10 mM Tris-Cl, pH 8.0, 2 mM EDTA, 0.1% SDS) and sonicated to 200-500 bp fragments using a Covaris S220 Focused-ultrasonicator. Sonicated chromatin was cleared by pelleting insoluble material at 13,000 RPM at 4°C followed by preclear with protein A/G beads and incubation with 1~2 ug antibody overnight (anti-Brg1, ab110641, Abcam; anti H3K27ac, ab4792, Abcam). Next, Protein A/G beads (20 ul) were added to the lysate and incubated at 4°C for 5 hrs. Immunoprecipitated material was washed with low salt buffer (20 mM Tris-Cl 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), and LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate) and one time with TE plus NaCl, followed by elution and reverse crosslinking overnight at 65°C. The quality controls of ChIPed DNA was performed with Qubit 2.0 Fluoremeter using dsDNA HS assay Kit (Q32854, ThermoFisher Scientific) and Agilent 2200 TapeStation System using Hightivity D1000 Reagenets (5067-5585, Agilent). The libraries for sequencing were prepared with the ThruPlex DNA-seq Kit (R400429, Rubicon Genomics) and sequenced on Illumina HiSeq 2500 system.
identifiers.namespace: GEO
identifiers.id: GSM3377376