|
| Status |
Public on Sep 30, 2024 |
| Title |
Xrn2 ChIP, auxin, replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
yeast
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell type: yeast
genotype: h- ura4-D18 ade6::ade6+ -Padh15 -skp1-OsTIR1-natMX6-P-adh15-skp1-AtTIR1-2NLS Spt5-3xmAID-5xFLAG-kanMX6 Xrn2-2xV5-hphMX
treatment: auxin treated
|
| Treatment protocol |
The cells were treated with either 1 mM auxin or DMSO for 2 hours at 25°C. Spike-in cells (S. cerevisiae SCC-6xV5) were added, and the cultures were crosslinked with formaldehyde, washed, and frozen.
|
| Growth protocol |
Strains were grown in YES media.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were disrupted using bead beating, and chromatin was isolated and sheared with a Bioruptor Pico. Immunoprecipitation (IP) was performed using beads coated with antibodies against Rpb1 (8WG16) or Xrn2 (anti-V5)
The library was prepared following the ChIP-Seq protocol. Briefly, IP DNA or input DNA was treated with pronase. The samples were then subjected to decrosslinking at 65°C, followed by RNA degradation using an RNase A/T1 mix and purification with the ChIP DNA Clean & Concentrator kit. Purified IP DNA or input DNA was used for library preparation according to the manufacturer's recommendations for the NEBNext Ultra II DNA Library Prep kit with Sample Purification Beads for Illumina.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Xrn2 ChIP with V5 antibody (Xrn2 wild-type)
|
| Data processing |
Reads trimmed and quality controlled with fastp (0.20.0)
Reads were aligned to concatenated genome of S.pombe and S. cerevisiae using STAR (2.7.3a)
Reads were split to each of the organisms keeping all alignments with samtools and bash command line
Reads which had too distant pairs were further filtered using a custom script
Aligned, filtered files were sorted and indexed using samtools
BigWig files were prepared using bamCoverage, normalised to sequencing depth while ignoring duplicates.
Input signal was subtracted from the ChIP signal. For DMSO-treated samples, a common input was used for background removal.
Reversal of the normalisation and application of spike-in normalisation factors were performed for comparisons of DMSO samples, while sequencing depth factors were used for comparisons between auxin- and DMSO-treated samples.
Negative values were filtered out.
Assembly: ASM294v2 from pombase.org
Supplementary files format and content: bigwig with normalised occupancy (input subtracted and without negative values). For comparisons between auxin and DMSO: sequencing depth normalisation was used (“normed” in filename). Comparing DMSO conditions spike-in was utilised (“spike” in filename). Additionally, mean bigwig file is provided.
|
| |
|
| Submission date |
Jul 30, 2024 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Krzysztof Kus |
| E-mail(s) |
krzysztof.kus@bioch.ox.ac.uk
|
| Phone |
07555185204
|
| Organization name |
University of Oxford
|
| Department |
Department of Biochemistry
|
| Lab |
Vasilieva Lab
|
| Street address |
South Parks Road
|
| City |
OXFORD |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL20584 |
| Series (1) |
| GSE273510 |
DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts. |
|
| Relations |
| BioSample |
SAMN42929992 |
| SRA |
SRX25521854 |
