|
| Status |
Public on Mar 07, 2018 |
| Title |
E14.5 male Tet1-KO PGC RRBS Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
E14.5 male Tet1-KO PGC
|
| Organism |
Mus musculus |
| Characteristics |
genetic background: Tet1-het GOF18∆PE-EGFP x Tet1-het GOF18∆PE-EGFP
genotype: Tet1-KO
age: E14.5
gender: male
cell type: PGC
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA from FACS-sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and DNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 26 µL final volume using the Savant SpeedVac Concentrator (Thermo) and following the manufacturer’s instructions.
Genomic DNA was digested by 20 units of MspI enzyme (NEB) in NEB buffer 2 at 37°C for 3 hrs, and digested DNA was purified using AMPure XP beads (Beckman-Coulter). Libraries were made following the NEBNext Ultra DNA Library Prep protocol, with the following modifications: 1) methylated adaptors were diluted 1:10 in nuclease free H2O to a final concentration of 1.5 µM; 2) following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma), and following the two step protocol according to the manufacturer’s specifications; and 3) PCR enrichment was carried out for 18 cycles using the KAPA Uracil+ DNA polymerase master mix (KAPA Biosystems) and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Pooled libraries were sequenced on the Illumina HiSeq 2000 instrument.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
MspI-digested reduced representation bisulphite sequencing of primordial germ cells
|
| Data processing |
Raw RRBS reads were first trimmed using Trim Galore (version 0.3.1, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with --rrbs parameter. Alignments were carried out to the mouse genome (mm9, NCBI build 37) with Bismark (version 0.13.0) with the -n 1 parameter. CpG methylation calls were extracted from the mapping output using the Bismark methylation extractor (version 0.13.0) with the –s --comprehensive --merge_non_CpG parameters. The number of methylated and unmethylated cytosines in a CpG context was extracted using bismark2bedGraph (default parameters).
Genome_build: mm9
Supplementary_files_format_and_content: Bismark cov file. Columns: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count unmethylated>
|
| |
|
| Submission date |
Jan 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Hill |
| E-mail(s) |
peter.hill1@imperial.ac.uk
|
| Organization name |
Imperial College London
|
| Street address |
Kensington
|
| City |
London |
| ZIP/Postal code |
SW7 2AZ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE76962 |
Epigenetic reprogramming enables the primordial germ cell-to-gonocyte transition (RRBS) |
| GSE76973 |
Epigenetic reprogramming enables the primordial germ cell-to-gonocyte transition |
|
| Relations |
| BioSample |
SAMN04421063 |
| SRA |
SRX1534381 |
