|
| Status |
Public on Sep 30, 2022 |
| Title |
AEP288 (qtr2∆) + preQ1-L1, small RNAs, - rRNA, IP-control, rep1 (Expt2) |
| Sample type |
SRA |
| |
|
| Source name |
AEP288 (qtr2{delta})
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: AEP288 (qtr2{delta})
genotype: FY7385; h- SPAC2F3.13c (qtr2)::NatMX leu1-32 ura4-D18 his3-D3
growth condition: growth in full medium (YES) at 30C
treatment: metabolic labeling with preQ1-L1 dervitave
experimental design: rRNA depletion and IP via strep-coated magnetic beads
|
| Growth protocol |
Strains AEP1 and AEP288 were grown in full medium (YES) with 0.1 µM preQ1-L1. Cultures were inoculated at an OD600 of 0.2 and grown to an optical density of 1.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Small RNAs were isolated using the PureLink™ miRNA Isolation Kit (Invitrogen) according to the manufacturer’s instructions. Ribosomal RNA depletion of 5S and 5.8S rRNA was achieved using reverse complementary oligonucleotides with a 5'-biotin tag as described before (Kraus et al. 2019). 8 µg small RNAs were used for hybridization with 100 µM oligonucleotides in 10 µL of formamide, 2.5 µL of 20 x SSC, 5 µL of 0.005 M EDTA, pH 8 with the following thermocycling: 80 °C for 5 min, ramp down to 25 °C at intervals of 5 °C per minute. The reaction was stopped with the addition of 2 µl RNAseOut ans 50 µl of 1 x SCC containing 20% formamide. Removal of rRNA/oligonucleotide hybrids was performed using DynabeadsTM MyOneTM Streptavidin C1 (ThermoFisher) according to the manufacturer’s instructions.
Library preparation of immunoprecipitated RNAs for deep sequencing was performed using the NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible; New England Biolabs) according to the manufacturer's protocols.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Q-RIP-Seq_exp2.csv
|
| Data processing |
Adapter-trimming was performed using Skewer version 0.2.2 and alignment to S. pombe RNAs (mRNA, ncRNA and tRNA; main and mitochondrial) from Pombase (https://www.pombase.org/) was achieved using Salmon version 14.0 and HISAT2 version 2.1.1, as a splice-site sensitive alignment program.
Conversion of sam to bam files was performed using SAMtools. Peak calling of aligned sequences was performed using the Bioconductor package exomePeak2 (adjusted P-value < 0.1).
Assembly: S.pombe genome version ASM294v2.29
Supplementary files format and content: comma-separated values files include read counts, log2-fold change, pvalues and the padj for IP (WT) and input (qtr2∆)
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| |
|
| Submission date |
Aug 03, 2022 |
| Last update date |
Sep 30, 2022 |
| Contact name |
Ann E. Ehrenhofer-Murray |
| Organization name |
Humboldt-Universität zu Berlin
|
| Department |
Institut für Biologie
|
| Street address |
Philippstr. 13
|
| City |
Berlin |
| ZIP/Postal code |
10099 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16192 |
| Series (1) |
| GSE210404 |
Identification of queuosine-modified RNAs in S. pombe using metabolic labelling with preQ1-L1 (Q-RIP-Seq) |
|
| Relations |
| BioSample |
SAMN30121666 |
| SRA |
SRX16812549 |
