|
| Status |
Public on Jul 27, 2021 |
| Title |
Sth1-AID degron induction RNA-Seq biological replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
yeast strains in small batch culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: FW7220 (MATa, his3D1, leu2D0, met15D0, ura3D0 LEU2::pGPD1-osTIR::LEU sth1::STH1-3V5-IAA7::KanMX6 rpb3::RPB3-3xFLAG::NATMX6) BY4741 derivative
treatment: 3-IAA
|
| Treatment protocol |
Cells were either left untreated (wild-type samples), or treated during exponential growth (OD600 ~0.8) by adding 3-indole-acetic acid (3-IAA, Sigma-Aldrich I3750) to a final concentration of 500 μM from a 1 M stock dissolved in DMSO, or an equivalent volume of DMSO (vehicle).
|
| Growth protocol |
Cells were grown in yeast extract-peptone-dextrose (YPD) medium in conical flasks at 30 °C, shaking at 300 RPM in incubators.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Together with fixed OD units of S. pombe cells as spike in, total RNA was extracted using the hot acid phenol protocol (acid phenol:chloroform:isoamyl alcohol 125:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in DEPC-treated sterile water.
Total RNA from yeast was incubated with rDNase (Machery-Nagel rDNase Set) and column purified (Machery-Nagel NucleoSpin RNA) prior to sequencing library preparation. Total RNA was used to generate strand-specific libraries for polyadenylated RNA sequencing using the TruSeq Stranded mRNA Library Prep Kit (Illumina RS-122-2101) according to the manufacturer’s instructions. Library quality control was performed using the Agilent 2200 TapeStation with D1000 screentape (Agilent Technologies). Each library was sequenced on the HiSeq 2500 platform using V4 chemistry (Illumina) and typically generated ~55 million 101 bp strand-specific paired-end reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Adapter trimming was performed with cutadapt (version 1.9.1) (Martin M, 2011) with parameters "--minimum-length=25 --quality-cutoff=20 -a AGATCGGAAGAGC -A AGATCGGAAGAGC".
BWA (version 0.5.9-r16) (Li & Durbin, 2009) using default parameters was used to perform the read mapping independently to both the S. cerevisiae (assembly R64-1-1, release 90) and S. pombe (assembly ASM294v2, release 44) genomes.
Genomic alignments were filtered to only include those that were primary, properly paired, uniquely mapped, not soft-clipped, maximum insert size of 2kb and fewer than 3 mismatches using BamTools (version 2.4.0; (Barnett et al, 2011)).
Read counts relative to protein coding genes were obtained using the featureCounts tool from the Subread package (version 1.5.1) (Liao et al, 2014). The parameters used were “-O --minOverlap 1 --nonSplitOnly --primary -s 2 -p -B -P -d 0 -D 1000 -C --donotsort”.
Genome_build: Ensembl R64-1-1 release 90
Supplementary_files_format_and_content: Tab-delimited text file containing raw counts generated by featureCounts where gene names are rows and columns represent all samples generated in the study.
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| |
|
| Submission date |
Jul 01, 2021 |
| Last update date |
Jul 27, 2021 |
| Contact name |
Folkert van Werven |
| E-mail(s) |
Folkert.vanWerven@crick.ac.uk
|
| Organization name |
Francis Crick Institute
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21656 |
| Series (2) |
| GSE179254 |
RSC mediated nucleosome positioning and GRFs form barriers in promoters to limit divergent noncoding transcription [RNA-Seq] |
| GSE179256 |
RSC mediated nucleosome positioning and GRFs form barriers in promoters to limit divergent noncoding transcription |
|
| Relations |
| BioSample |
SAMN19987985 |
| SRA |
SRX11325995 |
