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| Status |
Public on Sep 20, 2018 |
| Title |
Testis SSDS Sample 5 |
| Sample type |
SRA |
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| Source name |
Testis
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| Organism |
Mus musculus |
| Characteristics |
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)
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| Extracted molecule |
genomic DNA |
| Extraction 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalls performed using CASAVA version 1.8
Reads were aligned to the mm10 genome assembly. H3K4me3 ChIP-Seq reads were mapped to the reference genome using bwa aln (0.7.12).The pipeline described in Khil et al. Genome Res. 2012 was used to align SSDS reads to the genome.
Uniquely mapping fragments unambiguously derived from ssDNA (ssDNA type 1) and having both reads with a mapping quality score ≥ 30 were used for identifying hotspot locations (peak calling). NCIS was used to estimate the background fraction for each library. Peak calling was performed using MACS (v.2.1.0.20150420) with the following parameters : --ratio [output from NCIS] -g mm --bw 1000 --keep-dup all --slocal 5000. DSB hotspots within regions previously blacklisted were removed and hotspot strength was calculated as described previously(17).
Uniquely mapping reads with a mapping quality score ≥ 30 were used for peak calling. NCIS was used to estimate the background fraction relative to an input DNA library. Peak calling was performed using MACS (v.2.1.0.20150420) with the following parameters : --ratio [output from NCIS] -g mm --bw 1000 --keep-dup all --slocal 5000. Peak strength was subsequently calculated by subtracting the NCIS normalized input read count from the ChIP-Seq read count.
Genome_build: mm10
For each SSDS experiment, the associated BAM file contains all paired end ssDNA reads identified for that sample. Custom BAM tags describe properties of each read pair used for ssDNA detection:
it : ITR length
uh : microhomology length
os : microhomology offset
mm : # mismatches
For each SSDS experiment, a BED file of ssDNA fragments is also provided. Colum 4 gives the alignment phred score for each read (underscore separated). Column 5 gives the ITR-associated detail listed above (it_uh_os). DSB hotspots are provided in bedgraph format for each DMC1 SSDS sample. Background corrected hotspot strength is listed in the fourth column. DSB hotspots in blacklisted regions were not used for analyses.
For the Testis_H3K4me3_BSSeq sample, the processed data file is the bedgraph generated by bismark. This gives the percent methyated reads for each locus.
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| Submission date |
Jun 12, 2017 |
| Last update date |
Sep 20, 2018 |
| Contact name |
Kevin Brick |
| E-mail(s) |
brickkm@mail.nih.gov, kevbrick@gmail.com, brickkm@niddk.nih.gov
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| Organization name |
NIDDK
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| Department |
GBB
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| Street address |
5/205 Memorial Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE99921 |
Extensive sex differences at the initiation of meiotic recombination |
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| Relations |
| BioSample |
SAMN07222705 |
| SRA |
SRX2911853 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2664279_Testis_SSDS_T5.DSBhotspots.bedgraph.gz |
245.3 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM2664279_Testis_SSDS_T5.ssDNA_type1.bam |
2.0 Gb |
(ftp)(http) |
BAM |
| GSM2664279_Testis_SSDS_T5.ssDNA_type1.bed.gz |
79.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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