NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM3864896 Query DataSets for GSM3864896
Status Public on Feb 12, 2020
Title Z_1_combined
Sample type SRA
 
Source name Germ cells
Organism Mus musculus
Characteristics cell type: Germ cells
development stage: Zygotene spermatocytes (Z)
biology replication: Replication 1
genotype/variation: Wild type
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
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'.
 
Submission date Jun 10, 2019
Last update date Apr 20, 2021
Contact name Yuxuan Zheng
Organization name Fudan University
Street address 825 Zhangheng Rd.
City Shanghai
ZIP/Postal code 201203
Country China
 
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
Relations
BioSample SAMN12001379
SRA SRX6028061

Supplementary file Size Download File type/resource
GSM3864896_Z_1_combined_DNA_methylation.bed.gz 118.5 Mb (ftp)(http) BED
GSM3864896_Z_1_combined_chromatin_accessibility.bed.gz 928.9 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap