Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K27ac

Cell type

Cell type Class
Gonad
Cell type
Germline stem cells
NA
NA

Attributes by original data submitter

Sample

source_name
GS RA120h
cell type
Germline stem (GS) cell line
Sex
male
treatment
100 nM retinoic acid for 120 hours
antibody
anti-H3K27Ac (Active motif 39133)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For RAR ChIP, cells were fixed in 2 mM disuccinimidyl glutarate, 1.5 mM ethylene glycolbis succinimidyl succinate in PBS followed by 1% formaldehyde. Crosslinked cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris with protease inhibitors by sonication with Covaris S220. ChIP was performed by using anti-pan RAR antibody (Santa Cruz sc-773) and protein A/G agarose beads. For H3K4me3 and H3K27me3 ChIP, cells were fixed in 0.5% formaldehyde in PBS. Crosslinked cells were first lysed in 0.3M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mM Tris, 0.5 mM DTT, 0.5% TritonX-100 with protease inhibitors, then nuclei were pelleted by centrifugation of the above lysates on 1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mM Tris, 0.5 mM DTT. Nucleosome fractions were obtained by MNase treatment. ChIP was performed by using anti-H3K4me3 (Abcam ab8580) or anti-H3K27me3 (Upstate 07-449) and protein A/G agarose beads. For H3K27Ac ChIP, cells were fixed 1% formaldehyde in PBS. Crosslinked cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris with protease inhibitors by sonication with Covaris S220. ChIP was performed by using anti-H3K27Ac (Active motif 39133) and protein A/G agarose beads. Eluates were treated with RNase A and Proteinase K followed by phenol/chloroform extraction and ethanol precipitation of DNA.  For RAR, H3K4me3 and H3K27me3 ChIP, libraries were constructed from one (RAR) or five (H3K4me3 and H3K27me3) nanogram of ChIP DNA and five nanogram of input DNA using Illumina ChIP-seq sample prep kit (Illumina) and sequenced by GAIIX with Illumina Sequencing kit v4. For H3K27Ac ChIP-seq, libraries were constructed from one nanogram of ChIP DNA using TruSeq ChIP Sample Preparation Kit (Illumina) and sequenced by MiSeq with MiSeq Reagent Kit v2 (Illumina). 

Sequencing Platform

instrument_model
Illumina MiSeq

mm10

Number of total reads
10095465
Reads aligned (%)
95.8
Duplicates removed (%)
13.0
Number of peaks
1096 (qval < 1E-05)

mm9

Number of total reads
10095465
Reads aligned (%)
95.8
Duplicates removed (%)
12.9
Number of peaks
1060 (qval < 1E-05)

Base call quality data from DBCLS SRA