|
| Status |
Public on Nov 29, 2019 |
| Title |
ChIP-seq of Dhp1-GFP from swd22∆ cells |
| Sample type |
SRA |
| |
|
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: Dhp1-GFP in swd22delta cells
|
| Treatment protocol |
Genomic deletions of indicated genes in non-wild-type cells. Genomic C-terminal tagging with GFP of genes where indicated.
|
| Growth protocol |
Cultures were grown to logarithmic growth phase in rich media (YEA) at 18°C or 32°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde at room temperature for 20 minutes. Additional crosslinking with DMA were performed as needed for in each sample as indicated in each description section. Cells were lysed using bead-beater method. Lysates were sonicated and immunoprecipitations were performed in cold room conditions. Whole-cell extracts (WCE) were used as input control for each ChIP experiment. Reverse crosslinking was performed overnight at 65˚C, followed by treatment with RNase A and proteinase K. DNA was purified using QIAquick PCR purification kit (cat. no. 28014).
NEBNext Ultra II DNA Library Prep kit (NEB, E7645S) was used according to manufacturer's recommendation.
Libraries were analyzed using an Agilent 2100 BioAnalyzer (Agilent) and sequenced on the Illumina MiSeq or NextSeq 500 platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sample grown at 32°C, additional crosslinking was performed with DMA for 45 minutes at room temperature in addition to formaldehyde, and was sequenced on NextSeq 500 platform
Dhp1GFP_swd22∆ IP
|
| Data processing |
Alignment of adapter-trimmed reads were done using BWA-MEM.
For H3K9me ChIP-seq, reads per Genomic Content (RPGC) values were obtained using Deeptools suite. Correction for GC bias was assessed using input and the parameter--effectiveGenomeSize 12611000 in the correctGCbias module. Coverage was computed using the bamCoverage module and parameters --effectiveGenomeSize 12611000 --normalizeUsing RPGC --smoothLength 10 --binSize 1. Module bamCompare was used to compute input normalized enrichment values. For all other ChIP-seq coverage data, trimmed reads were aligned with BWA to the S. pombe v2.29 reference genome with defaults and the “-M” option for compatibility with Picard tools to produce BAM files. Aligned reads were deduplicated using Picard tools and used for normalization to reads per million (RPM) mapped. Normalized samples were then subject to input normalization by input subtraction from the immunoprecipitation sample. Bedtools was used to produce normalized coverage plot bedgraph files. Fold-enrichment bedgraphs were generated using MACS2.
Enrichment values are calculated for IP relative to whole-cell extract input control
For peak-calling, adapter-trimmed reads were aligned using STAR and normalized to reads per million (RPM). The locations of Dhp1 or Iss1-specific peaks were determined from normalized BedGraphs by identifying peaks in both the ChIP and input samples using an in-house script. Peaks that were found in both the ChIP and input BedGraphs were removed and the positions of the novel peaks that remained, those found only in the ChIP BedGraphs, were deemed to be Dhp1 or Iss1-specific.
Genome_build: asm294v2.29
Supplementary_files_format_and_content: bigWig and Bedgraph
|
| |
|
| Submission date |
Jun 17, 2019 |
| Last update date |
Nov 30, 2019 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL20584 |
| Series (2) |
| GSE123137 |
CPF is required for heterochromatin assembly and gene silencing [ChIP-seq] |
| GSE123144 |
CPF is required for heterochromatin assembly and gene silencing |
|
| Relations |
| BioSample |
SAMN12082721 |
| SRA |
SRX6081136 |
