alias: GSM4413853
title: GSM4413853: ChIPseq_6day_dox_EB_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq: 6-day dox-induced iPax7 mouse cells growing in monolayer were harvested by incubation with trypsin for 1-2 minutes. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + Complete-mini - Roche) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA + Complete-mini - Roche) for 10 minutes at +4°C. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + Complete-mini - Roche) and then sonicated. Cells were sonicated with a Branson sonicator at 18% power for 1 minute with intervals of 10 sec ON-10 sec OFF to achieve an average chromatin size of 300bp. After shearing, samples were centrifuged for 10 minutes at 16000g and snap frozen in liquid nitrogen if not processed immediately. For Pax7 ChIP, 25-40µg of chromatin (diluted to 500µl) were precleared for 4h at 4°C with 20µl of BSA-blocked Protein G-conjugated sepharose beads (GE healthcare). Samples were supplemented with 1/10 volume of 10% Triton X-100 and incubated overnight with anti-Pax7 (Developmental Studies Hybridoma Bank - Pax7) antibody. Immune complexes were recovered by incubation with 20µl of BSA-blocked Protein G-conjugated sepharose beads for 4h at 4°C and then washed 5 times with RIPA wash buffer (50mM HEPES KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% NP40, 0.25% Triton X-100, 0.7% Sodium Deoxycholate) and one time with TEN buffer (10mM TRIS HCl pH 8, 1mM EDTA, 50mM NaCl). Immunoprecipitated chromatin was recovered by incubating beads with 200µl of Elution buffer (50mM TRIS HCl pH 8, 10mM EDTA, 1% Sodium Dodecyl Sulfate) for 20 minutes at 65°C. Chromatin from IP and Input (equivalent to 1% of starting material) was reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM TRIS HCl pH 8, 1mM EDTA) supplemented with 4µl of RNaseA 20mg/ml and incubated for 2 hours at 37°C followed by Proteinase K treatment (4µl of 20mg/ml stock for each sample) for 30 minutes at 55°C. DNA was purified by Phenol-chloroform-isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3M Sodium Acetate pH 5 and 1.5µl of Glycogen and precipitated with 2 volumes of 100% Ethanol at -80°C for >1 hour. Followed 30 minutes centrifuge at 16000g, pellet were washed with 75% ethanol, air dried and dissolved in 45µl H2O. ChIP-seq libraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3'→5' exo-  NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5x Ampure XP beads and unbound DNAs were positively size selected by adding 0.4x Ampure XP beads (this step allows for retention of DNA fragments ranging 200-500bp). Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program. Libraries from iPax3 NIH3T3 cells were generated using the NEBNext DNA library prep kit (NEB).
identifiers.namespace: GEO
identifiers.id: GSM4413853