GSM4043225: Gata6 RA Input; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
G1/S-block cells treated with Retinoic Acid (RA)
tissue
Embryonic Stem Cell
genotype
KH2: 13 Flag-BAP-H3 tags
chip-antibody
none (input)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For native MNase chromatin preparation, nuclei were harvested from mESCs by hypotonic lysis TMSD buffer (40 mM Tris, 5 mM MgCl2, 0.25 M Sucrose with protease inhibitors), pelleted for 10 min at 3600 rpm at 4° C. Nuclei were solubilized by resuspending in NIB-250 (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose with protease inhibitors) containing 0.3% NP-40. A chromosome pellet was isolated by centrifuging the solubilized nuclei for 5 min at 600 rcf at 4° C. The chromosome pellet was washed with NIB-250 buffer, then resuspended with MNase digestion buffer (10 mM Hepes, 50 mM NaCl, 5 mM MgCl2, 5 mM CaCl2 with protease inhibitors) and treated with MNase (Sigma) until a DNA fragment size of 150-500 bp (1-3 nucleosomes) was attained. MNase digestion was stopped with 15 µl of EGTA (0.5 M) per 1 ml pellet and then spun at 600 rcf for 5 min at 4° C. The supernatant was placed in a fresh tube and the chromatin pellet was further processed by adding the same volume of BC500 (40 mM Tris, 5 mM MgCl2, 500 mM NaCl and 5% glycerol, and protease inhibitors) and EGTA (0.5 M) as in the MNase buffer plus EGTA step. The soluble chromosomes in BC500 and EGTA were then incubated for 30 min while rotating at 4° C, allowing for further enrichment of desired chromatin. Samples were pelleted at 600 rcf and the BC500 supernatant was pooled with an equal volume of MNase-treated supernatant to acquire the starting chromatin material for chromatin immunoprecipitations (IPs). For ChIP-seq, libraries were prepared by standard protocol. First, fragments are repaired to generate blunt 5' P ends using 'EndIT DNA End Repair kit' (Epicentre). Second, 3' A overhang was generated using 'Klenow Fragment (3´→ 5´ exo-)' (NEB). Third, Illumina barcodes were ligated using 'Rapid T4 DNA Ligase' (Enzymatics). Lastly, libraries were size selected using 'Ampure beads' (Beckman Coulter), then amplified, quantified and pooled. For RNA-seq, polyA-beads were used to purify mRNA followed by cDNA synthesis prior to standard library constructions as described above.