|
| Status |
Public on Oct 13, 2021 |
| Title |
Crispr Control ChIP-seq rep3 |
| Sample type |
SRA |
| |
|
| Source name |
N27_Crispr Control ChIP-seq
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: N27
cell type: dopaminergic neuronal cells
genotype/variation: wild type
chip antibody: H3K27ac(Abcam, ab4729, Lot # GR103379-1)
|
| Treatment protocol |
Cells were treated with rotenone or crispr knockout TFAM gene.
|
| Growth protocol |
N27 cells were routinely cultured in T175 flasks in RPMI medium (Gibco) supplemented with fetal bocine serum (FBS), L-glutamine, penicillin and streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNAs from treatment and control groups were isolated and quality controlled according to the Illumina protocols. A total of 2 μg RNA per sample was used as initial material for library construction. Chromatin immunoprecipitation (ChIP) was performed as described previously (Wang et al., 2 009b; Wang et al., 2 008). In brief, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 125 mM glycine. After chromatin fragmented to 100 to 1000bp by sonication, chromatin templates from 20 million cells were used for ChIP experiment. Samples were immunoprecipitated with 2- 4μg of H3K27ac antibody overnight at 4 °C followed by washing steps.
For RNAseq library construction, Poly-A containing mRNA molecules was purified by using poly-T oligo-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP, followed by second strand cDNA synthesis. These cDNA fragments then have the addition of a single ‘A’ base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. For ChIPseq library construction, after reverse cross-linking, ChIP-DNA fragments were purified and constructed into libraries by using the ThruPLEX DNA-seq Kit (Rubicon Genomics, Takara, Japan). Amplified libraries around 300 – 500 bp were isolated from agarose gel prior to sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
TFAMCK_ChIPseq_rep3
|
| Data processing |
The adapter sequences were removed from the raw sequencing data and the individual libraries were converted to the FASTA format.
Sequence reads were aligned to the rat genome (rn6) with HISAT2 (v2.1.0) and the resulting alignment files were reconstructed with the EdgeR3 package. RefSeq database (Build 37.3) was chosen as the annotation references for mRNA analysis.
The read counts of each transcript were normalized to the length of the individual transcript and to the total mapped fragment counts in each sample and expressed as reads per kilobase of exon per million fragments mapped (RPKM) of mRNAs in each sample.
ChIP-seq reads were mapped to the rat genome (rn6) using Bowtie aliger allowing up to two mismatches (Langmead, 2010). Only the uniquely mapped reads were used for further analysis. The EdgeR3 program was used to identify the differential peaks between treatment and control groups (cutoff: FC>2 and FDR<0.01).
SICER call peak program was used to call the peaks with a window size of 200 bp and a gap size of 400 bp, and an E value of 1.
The EdgeR3 program was used to identify the differential peaks between treatment and control groups (cutoff: FC>2 and FDR<0.01).
Genome_build: rn6
Supplementary_files_format_and_content: Tab-delimited text file include RPKM(Reads Per Kilobase Million) values were generated for RNA-seq samples and bigwig (.bw) files were generated for ChIP-seq samples.
|
| |
|
| Submission date |
Nov 18, 2019 |
| Last update date |
Oct 13, 2021 |
| Contact name |
Zhibin Wang |
| E-mail(s) |
zwang47@jhu.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Environmental Health and Engineering
|
| Street address |
615 N Wolf Steet
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL24688 |
| Series (1) |
| GSE140524 |
Mitochondrial Dysfunction Induces Epigenetic Dysregulation by H3K27 Hyperacetylation to Perturb Active Enhancers in Parkinson’s Disease Models |
|
| Relations |
| BioSample |
SAMN13318876 |
| SRA |
SRX7175028 |
