Antigen

Antigen Class

TFs and others

Antigen

Dmc1

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.

Sample

source_name

Testis

strain

C57BL/6J

age

Adult

genotype

wild-type

antibody

Anti-DMC1 Santa Cruz (C-20, sc 8973)

sequencing technique

ChIP-Single Stranded DNA Sequencing (SSDS)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Embryos were collected at E15.5, ovaries were dissected in cold PBS and stored at -80oC until use. 90 or 250 ovaries were fixed in 1 ml PBS with 1% paraformaldehyde for 3 min, quenched and homogenized with Dounce homogenizer. Cells were collected by 10 minute centrifugation at 900g using a bucket rotor. The pellet was washed in 1 ml of the following buffers: 1) PBS 2) 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH8. Cells were lysed in 0.5 ml of the lysis buffer (1% SDS, 10 mM EDTA, 50 mM TrisCl pH8 with complete protein inhibitor cocktail (Roche) and the chromatin was sheared with Misonix sonicator with the following parameters: efficiency 1, 10 s on, 20 s off, total sonication time 4 min. Chromatin was cleared by 10 min centrifugation at 12,000g at 4C. The supernatant was diluted 2-fold by ChiP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM TrisHCl , 167 mM NaCl) and dialyzed against the same buffer for 5 hour at 4oC. Chromatin was incubated with 6µg of custom made anti-DMC1 antibody and 20µl Dynabead beads (10002D, Invitrogen) at 4oC overnight followed by washing with 500µl of the following buffers: 1) 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM TrisHCl, 150 mM NaCl; 2) 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM TrisCl pH8, 500 mM NaCl; 3) 0.25 M LiCl, 1% Igepal, 1 mM EDTA, 10 mM TrisCl, pH8, 1% Deoxycholic acid. DNA protein complexes were eluted by two consecutive 15 min incubations at 650C using elution buffer (0.1M NaHCO3, 1%SDS, 5mM DTT). The eluates were combined and crosslinking was reversed at 650C for 5 hours. The samples were deproteinized and cleaned up with MinElute PCR purification kit (QIAGEN). The sequencing library was prepared as previously described (Brick et al. 2012). For H3K4me3 CHiP-Seq and DMC1 SSDS protocols from testis, testicular samples were obtained frozen and were directly thawed in 1% paraformaldehyde and gently dissociated. Testis sample preparation was performed as described previously (Brick et al., 2012). DMC1 and H3K4Me3 ChIP were performed as described previously (Smagulova et al., 2011; Khil et al., 2012; Brick et al., 2012) with minor modifications. Sequencing libraries for anti-H3K4Me3 samples were prepared according to manufacturer's protocol (Illumina) and anti-DMC1 libraries were prepared following the method described in (Khil et al., 2012).

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

183443278

Reads aligned (%)

51.1

Duplicates removed (%)

63.6

Number of peaks

6237 (qval < 1E-05)

mm9

Number of total reads

183443278

Reads aligned (%)

51.0

Duplicates removed (%)

63.8

Number of peaks

6221 (qval < 1E-05)