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| Status |
Public on Sep 09, 2020 |
| Title |
Primary spermatocytes 1 |
| Sample type |
SRA |
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| Source name |
Primary spermatocytes
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| Organism |
Mus musculus |
| Characteristics |
strain: FVB
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Spermatocytes and spermatids were isolated from testes using 25ug/mL liberase TM (Roche) followed by elutriation and percoll gradient centrifugation. Caput, corpus, cauda, and vas deferens were dissected and genomic DNA was isolated from all samples by lysing in extraction buffer (4.24 M guanidine thiocyanate (Sigma), 100 mM NaCl (Ambion), 1% N-Laurylsarcosine (Sigma), 150 mM DTT (Promega) and 200 ug/mL Proteinase K (Sigma)) at 56C for 2 hours followed by isopropanol precipitation. Samples were then sonicated on a Covaris S-220 to achieve a median size of 200-250 base pairs. DNA fragments were cleaned up using phenol:choloroform:isoamyl alcohol and phase-lock tubes. 500 ng of DNA was used as input for 2 rounds of sodium bisulfite conversion using the EZ DNA Methylation – Lightning Kit (Zymo).
Library construction was performed using TruSeq DNA Methylation Kit (Illumina) and Accel-NGS Methyl-Seq DNA Library Kit (Swift) according to the manufacturer’s instructions. For TruSeq (Illumina) libraries, 50ng bisulfite converted DNA was used and individual libraries were barcoded using the TruSeq DNA Methylation Index PCR Primers (IIlumina) to allow for multiplexed sequencing of libraries. Size selection and DNA purification was performed using Ampure XP beads (Agencourt) according to the TruSeq DNA Methylation kit protocol. For Accel-NGS Methyl-Seq (Swift) libraries, 30ng bisulfite converted DNA was used and barcoded following the (Swift) manufacturer’s instructions including 6 rounds of PCR. All final libraries were analyzed by Fragment Analyzer and quantitated using the Qubit as well as the KAPA Library Quantitation qPCR kit.
Bisulfite-Seq (Accel-NGS Methyl-Seq (Swift))
Bisulfite-Seq (Truseq)
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Libraries were sequenced on the Illumina HiSeq platform using the default RTA analysis software.
Swift BS-Seq sequences were trimmed with Trim Galore (v0.4.4; Cutadapt v1.9.1) with the options --clip_r1 12 --clip_r2 12, and Truseq libraries with the options --clip_r1 8 --clip_r2 8. Trimmed sequences were mapped to a personalised FVB/NJ mouse genome using Bismark (Krueger and Andrews, 2011) (v0.18.2). The personalised FVB/NJ genome was constructed by incorporating all high confidence SNPs from the latest VCF file released by the Mouse Genomes Project (mouse mgp.v5.merged.snps_all.dbSNP142.vcf; http://www.sanger.ac.uk/science/data/mouse-genomes-project) into the GRCm38 mouse reference using the genome preparation of SNPsplit (https://github.com/FelixKrueger/SNPsplit ; option --full_sequence). The CpG methylation calls were extracted and analysed using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
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| Submission date |
Jun 20, 2017 |
| Last update date |
Sep 09, 2020 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE100220 |
Cytosine methylation dynamics during spermatogenesis and post-testicular sperm maturation |
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| Relations |
| BioSample |
SAMN07257131 |
| SRA |
SRX2934503 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2674824_Swift_t5.cov.txt.gz |
217.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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