|
| Status |
Public on Jul 08, 2014 |
| Title |
MNase Sperm Supernatant 100 Units |
| Sample type |
SRA |
| |
|
| Source name |
Sperm, 100 units MNase, supernatant
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57/B6J
age: 10 weeks
tissue: sperm
chromatin preparation: Micrococcal Nuclease
micrococcal nuclease units: 100
fraction: supernatant
antibody: none
|
| Treatment protocol |
Sperm were isolated from the caudal epididymis and vas deferens of 10-week-old C57/6J mice by lacerating tissue in 1 mL of 37°C M2 medium (Sigma). Sperm were allowed to swim up for 1 hr, washed 1X in water, 1X PBS, crosslinked at 37°C with 1% formaldehyde for 15 min, quenched with 250mM glycine for 5 min and frozen in liquid nitrogen.
|
| Growth protocol |
E14 ES cells were cultured on gelatin-coated dishes without feeder cells in standard media containing serum and LIF at 37°C with 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Permeabilized ES and sperm nuclei were treated with 10 Units/10^6cells and 1 Unit/10^8cells of micrococcal nuclease (Worthington), respectively, for 5 min @ 37°C. Reaction was stopped with the addition of 10 mM EGTA on ice. For ES cells, nuclei were incubated for 4 hrs at 4°C with rotation, pelleted at 5000g for 5 min, and separated into supernatant and pellet (spun) or were treated the same without centrifugation and separation (unspun). After MNase digestion of sperm nuclei, digestion was centrifuged for 5000g for 5 min.
To characterize the entire range of MNase digestion products, we generated paired-end libraries of DNA fragments from both ES and sperm digestions and performed sequencing using Illumina HiSeq technology. Libraries were prepared as described in PMID: 22025700.
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Supernatant Fraction of DNA fragments resulting from Microccocal nuclease (100 Units) digestion of sperm chromatin.
|
| Data processing |
Removal of 3' adapter sequences using eautils 1.1.2.
Alignment of trimmed sequences to genome using Bowtie v2-2.2.2.
Creation of BigWig using standard scripts in Samtools v0.1.16 and bedGraphToBigWig (available at UCSC genome).
Tab-delimited txt files representing alignment of deepseq libraries to Transcriptional Start Site (TSS) in 20bp windows upstream and downstream of TSS 2000bp using the program SeqMiner v1.3.3e.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: BigWig, tab-delimited text files.
|
| |
|
| Submission date |
May 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Benjamin R Carone |
| E-mail(s) |
ben.carone@gmail.com
|
| Organization name |
UMass Medical School
|
| Department |
BMP
|
| Street address |
364 Plantation St
|
| City |
Worcester |
| State/province |
Massachusetts |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE58101 |
High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm |
|
| Relations |
| BioSample |
SAMN02803884 |
| SRA |
SRX555516 |
