GSM3770484: NS CHIP SF Gata4 rep1; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Gata4
Cell type
Cell type Class
Cardiovascular
Cell type
Cardiac fibroblasts
NA
NA
Attributes by original data submitter
Sample
source_name
neonatal mouse heart
strain
CD-1
genotype
alpha-MHC-GFP
cell line
Immortalized neonatal cardiac fibroblast cell line expressing large T antigen
exogenous retroviral expression
Gata4
chemical treatment
sb431542 and xav939
reprogramming time point
day 2
chip antibody
GATA-4 goat pAb (C-20); Santa Cruz Biotechnology; sc-1237x
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were crosslinked in 1% formaldehyde in suspension at room temperature for 10 minutes with gentle rotation. Crosslinking was quenched by addition of glycine (final 125 mM), followed by incubation at room temperature for 5 minutes with gentle rotation. Cell pellets were incubated in cell lysis buffer (20 mM Tris-HCl, pH 8, 85 mM KCl, 0.5% NP-40, protease inhibitors) for 10 minutes on a rotator at 4°C. Nuclei were isolated by centrifugation (2,500 x g, 5 minutes, 4°C), resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8, 1% SDS, protease inhibitors) and incubated on a rotator for 30 minutes at 4°C. Chromatin was sheared using a Covaris S2 sonicator for 15 minutes (60-second cycles, 5% duty cycle, 200 cycles/burst, intensity = 5) until DNA was in the 200–700 base-pair range. Chromatin was diluted five-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, protease inhibitors) and incubated with antibody (2 mg/million cells) at 4°C overnight under rotation. Antibodies used are Santa Cruz, Gata4, sc-1237x; Cell Signal Tech, Mef2c 5030; Santa Cruz, Tbx5 (C-20) sc-17866x. Antibody-protein complexes were immunoprecipitated using Pierce Protein A/G magnetic beads at 4°C for 2 hours under rotation. Beads were washed five times (2-minute washes under rotation) with cold RIPA buffer (50 mM HEPES- KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate), followed by one wash in cold final wash buffer (1xTE, 50 mM NaCl). Immunoprecipitated chromatin was eluted at 65°C with agitation for 30 minutes in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). High-salt buffer (250 mM Tris-HCl, pH 7.5, 32.5 mM EDTA, pH 8, 1.25 M NaCl) and Proteinase K were added and crosslinking was reversed by overnight incubation at 65°C. Samples were treated with RNase A and DNA was purified with Agencourt AMPure XP beads. Fragmented ChIP and input (whole-cell extract) DNA was end-repaired, 5′ phosphorylated and dA-tailed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645). Samples were ligated to adaptor oligos for multiplex sequencing (NEB E7335), PCR amplified, and sequenced on an Illumina NextSeq 500.