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| Status |
Public on Jan 09, 2023 |
| Title |
Slamseq_ski2_P_t0_r1 |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: yeast cells
genotype: ski2delta
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| Treatment protocol |
Cells were shifted to pre-warmed YPGal and then grown at 30°C for 3 hours (naïve). After this, cells are put in YPD for resting for 3 hours, cells were shifted back to prewarmed YPG for second induction (primed).
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| Growth protocol |
S. cerevisiae grown in YPD at 30°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with MasterPure RNA extraction kit from Lucigene The DNA was purified with phenol, precipitated with ethanol and sodium acetate at -20 °C, washed with cold 70% ethanol, and resuspended in 30 µl TE buffer.
SLAMSeq: Metabolic labeling of newly synthesized RNA molecules was done as previously described (Voichek et al., 2016). Briefly, 4-thiouracil (4tU) (Sigma) was dissolved in NaOH(83mM), Newly synthesized RNA was labeled for indicated time spans (10 min) at a final concentration of 5mM 4-thiouridine (4sU, Carbosynth). MES buffer with a final concentration 10mM PH 5.9 was added to media to avoid pH change as a result of NaOH addition. At each time points before harvesting, 4tU was added to media for ten minutes ahead of desired time point. cells were snap frozen in liquid nitrogen immediately at the time point required. Pulse and Chase: briefly, 4-thiouracil (4tU) (Sigma) was prepared as explained above. Prewarmed 4tU was added to culture media (YPD with MES buffer) containing wild-type cells. After 1 hour labelling with 4tU, cells were washed and resuspend in YPGal media and time points were collected at 0, 10 and 30 minutes (t0, t10, t30, t0’, t10’, t30’) by centrifugation and snap frozen in liquid nitrogen immediately. Between the first and second exposure to YPGal, cells were cultured for 3 hours in YPD. RNA purification was done with MasterPure Yeast RNA Purification Kit. Total RNA was subjected to thiol(SH)-linked alkylation by iodoacetamide (Sigma, 10 mM) at 50C for 15 minutes (Herzog et al., 2017), the reaction was stopped by 20 mM DTT and RNA was re-purified by ethanol precipitation. After rRNA depletion with RiboPool Depletion Kit, library was prepared by Ultra™ II Directional RNA Library Prep Kit for Illumina® and sequenced single end with read length 150bp on Nextseq 500 Illumina sequencer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
ski2∆ parental strain background: BY4741(MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0)
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| Data processing |
F SLAMseq data was anaysed with slamdunk provided by nextflow pipeline(v 1.0.0). https://nf-co.re/slamseq . As stranded library was prepared with dUTP method and fastq files were first converted to reverse complementary reads to feed into slamdunk nf core pipeline. Adapter contamination and low quality region was trimmed with TrimGalore, trim length 30bp was used instead of default value. When aligned to genome, transcript annotation downloaded from SGD database (version 64-1-1) (Xu et al., 2009) were first converted into bed file and then used as input for parameter utrbed. At least 2 conversions cooccur in one reads is regarded as a confident call for nascent RNA. SNP masking was employed to uncouple Single Nucletide Polymorphism from converted nucleotides.
Assembly: V64-1-1
Supplementary files format and content: bigwig format of spike in normalised reads
Supplementary files format and content: bigwig format of spike in normalised nascent reads
Library strategy: SLAMseq
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| Submission date |
Nov 20, 2022 |
| Last update date |
Jan 09, 2023 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
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| Organization name |
ScilifeLab - Karolinska Institutet
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| Department |
MTC
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| Street address |
Nobels väg 16
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| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
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| Platform ID |
GPL31112 |
| Series (1) |
| GSE218400 |
Measurement of mRNA stability during transcriptional memory in mutants for RNA degradation |
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| Relations |
| BioSample |
SAMN31806563 |
| SRA |
SRX18322705 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6744273_ski2_P_t0_r1_S10.crick.bw |
12.9 Mb |
(ftp)(http) |
BW |
| GSM6744273_ski2_P_t0_r1_S10.nascent.crick.bw |
6.6 Mb |
(ftp)(http) |
BW |
| GSM6744273_ski2_P_t0_r1_S10.nascent.watson.bw |
6.5 Mb |
(ftp)(http) |
BW |
| GSM6744273_ski2_P_t0_r1_S10.watson.bw |
12.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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