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| Status |
Public on Jun 03, 2020 |
| Title |
Round Spermatids-H3K4me3 |
| Sample type |
SRA |
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| Source name |
testis
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| Organism |
Rattus norvegicus |
| Characteristics |
cell type: round spermatids
strain: Sprague-Dawley
chip antibody: mouse anti-histone H3 (tri methyl K4) antibody (2 μg/IP) (ab12209, Abcam, UK)
treatment: no treatment
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Round spermatid cell lysates were prepared from nuclei by sonication and specific Histone modification antibodies were used for histone-DNA chromatin immunoprecipitation (ChIP) assays using the SimpleChIP Enzymatic Chromatin IP kit (9003, CST, USA) according to the protocol provided by the manufacturer.
Immunoprecipitated DNA fragments were subjected to library preparation using TrueSeq DNA Kit (FC-121-2001, Illumina, USA). NEB Next index set 3 was used for barcoding and adapter ligation (E7710S, NEB, USA). Briefly, 5 – 10 ng of ChIP DNA was subjected to end repair which converts the overhangs resulted from fragmentation into blunt ends using an end repair mix. This end repair mix has 3' to 5' exonuclease activity which removes the 3' overhangs and the polymerase activity fills in the 5' overhangs. After this step, a single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments which prevent the end repaired fragments from ligating to each other which is called as A tailing. Following this step, adapter ligation was performed where adapters having indexes were ligated to the ends of the DNA fragments, enabling them for hybridization onto a flow cell. Purification of adapter ligated fragments followed by enrichment of library was done to amplify DNA fragments having adapter sequences. DNA sequencing was performed in Illumina NextSeq 500 platform (Illumina Inc, USA) with a read length of 75 bp and paired end chemistry.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The ChIP-seq reads obtained from the sequencer were demultiplexed using bcl2fastq tool. Quality and adapter trimming was done using FastQC software.
The processed data/reads were then aligned with rat genomic assembly rn6 by using Bowtie 1.2 (parameters -m 1 -best).
Peak calling was done using MACS1.4 with p-value threshold of 0.001 and 10% of FDR cut-off.
Genome_build: Rn6
Supplementary_files_format_and_content: bed files
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| Submission date |
Jun 02, 2020 |
| Last update date |
Jun 06, 2020 |
| Contact name |
Singh Rajender |
| E-mail(s) |
[email protected]
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| Organization name |
Central Drug Research Institute
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| Street address |
Lucknow
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| City |
Lucknow |
| State/province |
Uttar Pradesh |
| ZIP/Postal code |
226021 |
| Country |
India |
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| Platform ID |
GPL20084 |
| Series (1) |
| GSE151608 |
Histone methylation regulates gene expression in the round spermatids to set the RNA payloads of sperm |
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| Relations |
| BioSample |
SAMN15080948 |
| SRA |
SRX8452881 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4586560_H3K4_Negative_peaks.bed.gz |
108.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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