|
| Status |
Public on Dec 22, 2022 |
| Title |
red1W298A/F305A-FTP direct RNA |
| Sample type |
SRA |
| |
|
| Source name |
S. pombe grown in YEA liquid culture to an OD600 of around 2
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain number: F3271
genotype: MatMsmt0, leu1-32, ura4-D18, ade6-M210, red1W298A/F305A-3xFTP::hyg
|
| 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 |
total RNA |
| Extraction protocol |
RIP-RNA: Cell pellets that were snap-frozen and ground was resuspended in purification buffer and the clarified supernatants prepared post centrifugation were incubated with IgG beads. After IgG binding TEV cleavage was performed using AcTEV protease and the eluate collected was incubated with anti-Flag beads. Flag bound proteins were subsequently eluted using flag peptide and two third of the flag eluate was taken for DNase treatment and the RNA was purified using RNA Clean and Concentrator Kit.
Direct RNA seq.: 200ng of RIP-purified RNA was used for library preparation using Oxford Nanopore direct RNA sequencing library kit (SQK-RNA002; Version: DRS_9080_v2_revM_14Ag2019) protocol. The prepared libraries were run on individual flow cells following manufacturer guidelines.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
RIP-purified RNA was used for direct RNA seq. using ONT.
|
| Data processing |
Nanopore reads were aligned with minimap (2.10) with long-read spiced alignment (with splice and -k7 parameters).
Strand-specific bigwig tracks were generated using bamCoverage to the pombe genome (ASM294v2) with the parameter ‘—binSize 1’.
Genome_build: ASM294v2
Bigwig tracks were generated after normalisation by the sum of the raw signals of chromosome I and II multiplied by 100 million.
|
| |
|
| Submission date |
Jun 14, 2022 |
| Last update date |
Dec 24, 2022 |
| Contact name |
Attila Horvath |
| E-mail(s) |
attila.horvath@anu.edu.au
|
| Organization name |
The Australian National University
|
| Street address |
Garran Road 131
|
| City |
Canberra |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL31123 |
| 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 |
SAMN29043995 |
| SRA |
SRX15703928 |
