Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Gonad
Cell type
Testis
MeSH Description
The male gonad containing two functional parts: the SEMINIFEROUS TUBULES for the production and transport of male germ cells (SPERMATOGENESIS) and the interstitial compartment containing LEYDIG CELLS that produce ANDROGENS.

Attributes by original data submitter

Sample

source_name
H3f3b null testis tissue
cell type
Adult Mouse Testis tissue
strain
C57BL/6
genotype/variation
H3f3b null
chip antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin Preparation: Testes were initially isolated and stored at -80C. Tissue was tehn chopped and placed in a 15ml conical with DMEM. 37% formaldehyde was added toa final concentration of 1% and rocked for 12 minutes. 1.25 ml of glycine was added, rocked for 2 minutes then centrifuged. Tissue was then washed three times with 10ml sterile PBS. 1 ml cell lysis buffer(5 mM PIPES pH8, 85 mM KCl. Add igepal fresh each time to give a final concentration of 1% (10 μl/ml) plus protease inhibitors (Roche – 11 836 170 001)) was added and incubated for 15 min. Cells were then dounced 23 times, transferred to 1.5 ml tube and spun for 5 min at 2500G. Cells were then re-suspended in 200ul nuclear lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, and 1% (w/v) SDS plus protease inhibitor) and incubated on ice for 30 minutes. Pellet was flash frozen in LN2, thawed slightly and sonicated (15 minutes, 30 seconds on, 1 minute off). PCR was used to check fragment size. Chromatin was diluted with IP dilution buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% (v/v) igepal, 0.25% (w/v) deoxycholic acid, 1 mM EDTA pH8 plus protease inhibitors). Aliquots of each sample were taken and incubated with one of the following antibodies overnight at 4 degrees C: 3ul of anti trimethyl histone H3 Lys 4 (H3K4me3, Millipore # 04-745). 15ul of each sample was also saved for input. Samples were incubated with antibody overnight at 4 degrees C. 15 ul magnetic Protein G beads (Cell Signaling 9006S) were added to each sample and incubated for 2 hours at 4. Beads were magnetized and washed 2x in IP dilution buffer, 1x in IP wash buffer 2 (100 mM Tris-HCl pH 9, 500 mM LiCl, 1% (v/v) igepal, 1% (w/v) deoxycholic acid), 2x in IP wash buffer 3 (100 mM Tris-HCl pH 9, 500 mM LiCl, 150 mM NaCl, 1% (v/v) igepal, 1% (w/v) deoxycholic acid). Chromatin was eluted with 100ul elution buffer (50 mM NaHCO3, 1% (w/v) SDS) and vortexed at RT for 50 min. Beads were magnetized, supernatant (including input) was incubated at 67 degrees C O/N to reverse crosslinks. Qiagen's QiaQuick PCR purification Kit (Qiagen 28104) was used to purify DNA, and RT-PCR and qPCR was used to determine specificty of chromatin. Libraries were prepared according to directions stated O'geen et al. 2011 (PMID: 21913086)

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
12535028
Reads aligned (%)
97.7
Duplicates removed (%)
10.8
Number of peaks
386 (qval < 1E-05)

mm9

Number of total reads
12535028
Reads aligned (%)
97.4
Duplicates removed (%)
11.0
Number of peaks
381 (qval < 1E-05)

Base call quality data from DBCLS SRA