|
| Status |
Public on Jul 26, 2018 |
| Title |
TGBS_swim-up sperm_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Spermatozoa
|
| Organism |
Mus musculus |
| Characteristics |
sperm fraction: Swim-up sperm
strain: C57BL/6
dna selection: SureSelect (Agilent)
|
| Treatment protocol |
After mild sonication to separate sperm head from the tail, HRCS (Histone Replacement- Complete Sperm) was isolated from total sperm fraction by 82% Percoll precipitation.
|
| Growth protocol |
Caudal epididymis and vas deferens were incubated in M2 medium for 1 hour at 37C to prepare total sperm fraction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After treatment of sperm lysis buffer, genomic DNA was extracted from each sperm sample. DNA is purified by RNase and pronaseK treatment, and extracted by PCI separation and EtOH precipitation.
Libraries for targeted methylome sequencing were prepared according to the post-bisulfite adaptor tagging (PBAT) protocol (Miura F et al., 2012; Miura F and Ito T, 2015) with slight modifications. Briefly, 300 ng of genomic DNA was fragmented to ~500 bp with Covaris S220 and used for target enrichment with SureSelectXT Mouse Methyl-Seq kit (Agilent). The enriched DNA was subjected to PBAT followed by 5 cycles of PCR enrichment with Kapa Library Amplification Kit. Single-read sequencing (100 cycles) was performed on Illumina HiSeq 2500 using HiSeq SR Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2. Two indexed libraries were combined with a high concentration of spike-in PhiX control DNA (20%) and loaded on a single lane of the flow cell.
Target BS-seq
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina Casava1.4 software used for basecalling.
Targeted methylome sequencing reads were mapped to the mouse reference genome sequence (mm9) using Bmap (http://itolab.med.kyushu-u.ac.jp/BMap/index.html).
Methylation levels were calculated for individual CG sites.
Genome_build: mm9
Supplementary_files_format_and_content: BED files include chrmosome, position, strand, the number of mC reads and the number of total reads.
|
| |
|
| Submission date |
Apr 13, 2018 |
| Last update date |
Jul 26, 2018 |
| Contact name |
Hiromitsu Araki |
| E-mail(s) |
araki.hiromitsu.596@m.kyushu-u.ac.jp
|
| Organization name |
Kyushu University
|
| Street address |
744 Motooka Nishi-Ku
|
| City |
Fukuoka |
| ZIP/Postal code |
819-0395 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE113137 |
DNA methylation analysis for target regions in swim-up sperm and HRCS. |
| GSE113150 |
Mapping of histone-binding sites in histone replacement-completed spermatozoa |
|
| Relations |
| BioSample |
SAMN08930496 |
| SRA |
SRX3934596 |
