|
| Status |
Public on Mar 17, 2020 |
| Title |
GCERA |
| Sample type |
SRA |
| |
|
| Source name |
Male germ cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Holtzman strain
age: 75 - 90 days old
chip antibody: Anti-ERα (Millipore, CS200620, lot DAM1794327)
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
The animals were sacrificed and testes were dissected out, detunicated and the tubules were teased apart. Interstitial cells were lysed by hypotonic treatment by suspending the tubules in distilled water twice for 1 min and decanting the supernatant. The tubules were then resuspended in DMEM F12 and treated with trypsin followed by DNase twice and incubated at 37ᵒC on the shaker (80rpm) for 8 mins and 5 mins respectively. The tubules were then allowed to settle down and the supernatant was passed through 70µm nylon mesh. The filtrate collected through the 70µm nylon mesh was filtered through 40µm nylon mesh to separate Sertoli cells. The filtrate thus obtained was centrifuged at 800rpm for 8 mins at 4ᵒC. The supernatant was discarded and the pellet was resuspended in Hank’s balanced salt solution (HBSS). A part of the pellet was separated for RNA extraction to check for purity of the germ cell enrichment and the remaining cells were crossed-linked using formaldehyde and stored at -80ᵒC for chromatin immunoprecipitation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The enriched germ cells were subjected to sonication followed by immunoprecipitation using ERα and ERβ antibody.
The immuno-precipitated DNA was quantified by Qubit2.0 and for size distribution Agilent Bioanalyser was used as a QC step for chip sequencing library. Chip Sequencing libraries were generated using NEBNext® Ultra™ II DNA kit for Illumina as per the recommended protocol. Briefly, a total amount of 200-500pg input material is used for Chip sequencing. The fragmented DNA overhangs were converted into blunt ends via exonuclease/polymerase activities (END REPAIR). After adenylation of 3’ ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated adapter molecules on both ends were selectively enriched in final PCR reaction. Products were purified using AMPure XP system (Beckman Coulter, Beverly,USA) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system for size distribution. The final libraries are quantified using Kapa qPCR Kit for adapter ligated molecules and was sequenced on PE75 Illumina platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Alignment was done using Bowtie version 1.2 to reference genome Rnor06
Peaking calling was done using MACS version 14
Peaks with the following threshold were considered: p Value <0.01; Fold enrichment >2
Genome_build: Rnor6
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Mar 16, 2020 |
| Last update date |
Mar 17, 2020 |
| Contact name |
Nafisa Balasinor |
| E-mail(s) |
[email protected]
|
| Organization name |
ICMR-National Institute for Research in Reproductive Health (NIRRH-ICMR)
|
| Department |
Neuroendocrinology
|
| Street address |
Jehangir Merwanji Street
|
| City |
Mumbai |
| State/province |
Maharashtra |
| ZIP/Postal code |
400012 |
| Country |
India |
| |
|
| Platform ID |
GPL11282 |
| Series (1) |
| GSE147079 |
Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells |
|
| Relations |
| BioSample |
SAMN14387545 |
| SRA |
SRX7922284 |
