|
| Status |
Public on Nov 26, 2020 |
| Title |
Input DNA |
| Sample type |
SRA |
| |
|
| Source name |
testicular cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
age: 35-50days
tissue: testis
cell type: round/Elongating spermatids
chip antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were prepared from 35-50days old rat testis, decondensed and chromatin immunoprecipitation was done using anti-H1T2 antibodies. Immunocomplexes were captured on 50 μl protein A Dynabeads. Beads with bound immunocomplexes were washed, eluted and reverse cross-linked at 65°C overnight and DNA was recovered using Qiagen PCR purification kit. Input was prepared with 5% of the sonicated chromatin.
Library DNA was purified using Qiagen DNA purification kit. Sequencing was performed with Illumina HiSeq X Ten system, and raw sequence reads containing adaptor sequences were trimmed and filtered with Phred quality threshold as 20. Read pairs falling below 20 bp after clipping were removed
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
Input_1
|
| Data processing |
Quality Control Analysis and modification were performed in a three step procedure. Firstly, FASTQC was used to extract the sequence quality measures from the raw files. Secondly, Adaptor clipping was done with a stringent overlap condition of 1bp using trim_galore.The pairs in which this step resulted in reduction of sequence length to below 20bp were removed. Finally, pairs where read quality was found to be less than 20 were removed.
Bowtie2 was used to align the paired end reads for each sample. For this purpose UCSC Rattus norvegicus genome (rn6) was used as a reference. SAM files were converted to respective BAM files using SAMtools, which were subsequently sorted and filtered off duplicates. Resulting processed BAM files were the passed through another quality check using BAMQC. BED files were prepared with bedtools for visualisation and peak identification corresponding to each BAM file. For both the conditions (treated and control/input) replicates were merged using SAMtools merge
Peak identification was done using macs2 for the mergerd treated BAM against the merged input/control sample as control. p-value 1e-05 was used as cutoff.
Peak reigions were annotated to find basic annotations related to the overlaps using Homer
Selected regions with high enriched peaks were validated using ChIP-PCR
Genome_build: rn6
Supplementary_files_format_and_content: H1T2_peaks
|
| |
|
| Submission date |
Nov 25, 2020 |
| Last update date |
Nov 26, 2020 |
| Contact name |
Manchanahalli Rangaswamy Satyanarayana Rao |
| E-mail(s) |
mrsrao@jncasr.ac.in
|
| Phone |
9108871102
|
| Organization name |
Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR)
|
| Department |
Molecular Biology and Genetics Unit (MBGU)
|
| Lab |
Chromatin Biology Lab (CBL)
|
| Street address |
Jakkur, Bengaluru, Karnataka
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560064 |
| Country |
India |
| |
|
| Platform ID |
GPL24688 |
| Series (1) |
| GSE162144 |
Genome-wide occupancy of linker histone variant H1T2 in round/elongating spermatids of rats |
|
| Relations |
| BioSample |
SAMN16912466 |
| SRA |
SRX9582469 |
