Antigen

Antigen Class

TFs and others

Antigen

Dmc1

Cell type

Cell type Class

Gonad

Cell type

Ovary

MeSH Description

The reproductive organ (GONADS) in female animals. In vertebrates, the ovary contains two functional parts: the OVARIAN FOLLICLE for the production of female germ cells (OOGENESIS); and the endocrine cells (GRANULOSA CELLS; THECA CELLS; and LUTEAL CELLS) for the production of ESTROGENS and PROGESTERONE.

Sample

source_name

Ovary

strain

C57BL/6J

age

15.5 days post coitum

genotype

wild-type

antibody

Anti-DMC1 GP1 custom

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

132726861

Reads aligned (%)

38.6

Duplicates removed (%)

72.6

Number of peaks

6876 (qval < 1E-05)

mm9

Number of total reads

132726861

Reads aligned (%)

38.6

Duplicates removed (%)

72.7

Number of peaks

6868 (qval < 1E-05)