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| Status |
Public on Feb 12, 2020 |
| Title |
pL_2_combined |
| Sample type |
SRA |
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| Source name |
Germ cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Germ cells
development stage: Preleptotene spermatocytes (pL)
biology replication: Replication 2
genotype/variation: Wild type
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 1,000 cells were transferred into a 0.2 μL PCR tube containing 7 μL of ice-cold lysate buffer (50 mM Tris-HCl (pH7.4), 50 mM NaCl, 0.25 mM EDTA, 10 mM DTT, 0.25 mM PMSF and 0.5% NP-40, plus 2 pg λDNA). After gently vortex, the cell lysate was kept on ice for 10 min. The GpC methyltransferase M. CviPI and SAM were then added to the lysate to a final volume of 10 μL containing 1 U/μL M. CviPI and 160 μM SAM. The in vitro methylation of nuclei was performed by incubating the mixture in a thermocycler at 37℃ for 45 min followed by heating at 65℃ for 25 min to inactive the enzyme activity. After in vitro methylation, 1 μL of 20 mg/mL protease (Qiagen) was added and the mixture was incubated for 3 hrs at 50℃ to release genomic DNA. The released genomic DNA was then bisulfite converted using the EZ-96 DNA Methylation-Direct MagPrep (Zymo) according the manufacturer’s instructions. Afterwards, the purified DNAs were annealed using random nonamer primers with a 5’-biotin tag (5′-Biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′) in the presence of Klenow fragments (3’-5’ exo-, NEB). Then, the primers were digested by exonuclease I (NEB) and the DNA was purified using Agencourt Ampure XP beads (Beckman Coulter). Dynabeads M-280 Streptavidin (Invitrogen) were then used to immobilize the newly synthesized biotin-tagged DNA strands, and the original bisulfite-converted DNA templates were removed. Second DNA strands were synthesized using Klenow fragment with random nonamer primers (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′). After washing, the beads were used to amplify libraries using 15 cycles of PCR with the universal primer and index primer (NEB). The amplified libraries were purified with Agencourt Ampure XP beads twice. Fragment from 300 bp to 800 bp were selected by argarose gel electrophoresis and purified by Zymoclean Gel DNA Recovery Kit (Zymo). Finally, libraries were pooled and sequenced on the Illumina HiSeq 2500 sequencer for 150-bp paired-end sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: COOL-seq
Raw COOL-seq sequencing reads were firstly trimmed 9-bp random primer, removed adaptors and low-quality bases using trim_galore (version: 0.1.3) with parameters ‘--quality 20 --stringency 3 --length 50 --clip_R1 9 --clip_R2 9 --paired --trim1 --phred33’. Clean reads were mapped against human reference genome hg19 (UCSC) using Bismark (version: 0.7.6) with a paired-end and non-directional mode (parameters ‘--fastq --non_directional –unmapped --phred33-quals’)1. For improving the number of mapped reads, the unmapped reads were realigned again the same reference genome in a single-end aligned mode. After alignment, PCR duplicated reads were removed using SAMtools (version: 0.1.18). We defined the methylation level of each detected cytosine site as the methylated reads ‘C’ dividing by the all detected reads (methylated and unmethylated reads, ‘C+T’). We used WCG (ACG/TCG) for DNA methylation analysis and GCH (GCA/GCC/GCT) for chromatin accessibility analysis.
Genome_build: Mus musculus (mm10)
Supplementary_files_format_and_content: For either 'DNA_methylation' or 'chromatin accessibility' data, each column indicates 'chr, start, end, unmethylated C reads, methylated C reads'.
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| Submission date |
Jun 10, 2019 |
| Last update date |
Apr 20, 2021 |
| Contact name |
Yuxuan Zheng |
| Organization name |
Fudan University
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| Street address |
825 Zhangheng Rd.
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| City |
Shanghai |
| ZIP/Postal code |
201203 |
| Country |
China |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE132446 |
Refined spatial temporal epigenomic profiling reveals intrinsic connection between PRDM9-mediated H3K4me3 and the fate of double-stranded breaks |
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| Relations |
| BioSample |
SAMN12001382 |
| SRA |
SRX6028058 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3864893_PL_2_combined_chromatin_accessibility.bed.gz |
906.2 Mb |
(ftp)(http) |
BED |
| GSM3864893_pL_2_combined_DNA_methylation.bed.gz |
113.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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