|
| Status |
Public on Dec 22, 2022 |
| Title |
red1288-345DEL K9 ChIP Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
S. pombe grown in YEA liquid culture to an OD600 of 0.5-0.8
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain number: F3436
genotype: MatMsmt0, leu1-32, ura4-D18, ade6-M210, red1288-345DEL :: natNT2 (+3'UTR)
antibody: H3K9me2
|
| Treatment protocol |
Samples for ChIP seq. was treated using formaldehyde solution to a final concentration of 1% at room temperature (RT) for 15 min and quenched by addition of glycine to a final concentration of 150 mM for 5 min at RT
|
| Growth protocol |
S. pombe strains used for the study were grown in YEA liquid culture to an OD of 0.8 - 2.2 according to experiment performed.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP DNA: The collected lysate was used for chromatin shearing to a median size of ~300bp by sonication using Covaris S2. The samples were then incubated overnight with H3K9me2 antibody (mAbcam 1220) followed by an incubation with an appropriate bead slurry (nProtein A Sepharose, GE Healthcare, 5280-01). Post washing , the bound protein and chromatin fractions were eluted using elution buffer. The samples were then decrosslinked and the DNA was purified using Phenol: Chloroform: Isomyl alcohol method following RNase treatment.
ChIP seq: The purified DNA pellet was used for library preparation using NEBNext Ultra II DNA library prep kit for Illumina (NEB, E7645) following manufacturer instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
H3K9me2 ChIP DNA was directly used for library preparation
|
| Data processing |
Paired-end illumina reads were subjected to a thorough Quality Control (QC) and the FastQC 0.11.8 was used to generate QC reports. Low complexity regions were filtered out with prinseq-lite.pl from PRINSEQ-lite 0.20.4 (maximum dust was set to 3) and the remaining sequences were trimmed using Trimmomatic (SLIDINGWINDOW: 4 nt; phred quality cut-off: 15, MINLEN=36).
QC filtered were aligned with BWA MEM -0.7.17. Aligned bam files were sorted and indexed using samtools 1.3.1.
Potential PCR duplications were removed with samtools using the rmdup option
Genome_build: ASM294v2
Bigwig tracks were generated using bamCoverage to the pombe genome (ASM294v2) with the parameter “—binSize 1”. Bigwig tracks were normalised by the sum of the raw signals of chromosome I and II multiplied by 100 million. Further, bigwig tracks of a dusty filtering of 4 was used for further analysis and screenshots.
|
| |
|
| Submission date |
Jun 14, 2022 |
| Last update date |
Dec 24, 2022 |
| Contact name |
Attila Horvath |
| E-mail(s) |
[email protected]
|
| Organization name |
The Australian National University
|
| Street address |
Garran Road 131
|
| City |
Canberra |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL28961 |
| Series (1) |
| GSE206106 |
Mechanistic insights into the role of the canonical poly(A) polymerase Pla1 in RNA surveillance by the fission yeast MTREC complex |
|
| Relations |
| BioSample |
SAMN29044003 |
| SRA |
SRX15703920 |
