|
| Status |
Public on Dec 20, 2018 |
| Title |
Rap1-AID degron no induction total RNA-Seq biological replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
yeast strains in small batch culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: FW3877 (MATa leu2Δ0 met15Δ0 ura3Δ0 rap1::RAP1-3V5-IAA7::KANMX6 his3::pGPD1-osTIR::HIS3) BY4741 derivative
treatment: DMSO (30min)
|
| 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. 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. For total RNA-Seq libraries, intact yeast total RNA was depleted of rRNA with the Illumina RiboZero Gold rRNA removal kit (yeast) (Illumina MRZY1324). 1 μg of yeast total RNA was used to generate strand-specific libraries for total RNA sequencing using the TruSeq Stranded Total RNA Library Prep Kit (Illumina RS-122-2202) according to the manufacturer’s instructions. For polyA RNA-Seq libraries, 500 ng of intact yeast 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 2500 |
| |
|
| Description |
allSamples_EXP1_RNASeq_RSEM_TPM.tsv.gz
RAP1_TOTAL_UNINDUCED_R3
|
| 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".
The RSEM package (version 1.3.0) (Li and Dewey, 2011) in conjunction with the STAR alignment algorithm (version 2.5.2a) (Dobin et al., 2013) was used for the mapping and subsequent gene-level counting of the sequenced reads with respect to all S. cerevisiae genes downloaded from the Ensembl genome browser (assembly R64-1-1, release 90; Kersey et al., 2016). The parameters used were "--star-output-genome-bam --forward-prob 0", and all other parameters were kept as default.
Differential expression analysis was performed with the DESeq2 package (version 1.12.3) (Love et al., 2014) within the R programming environment (version 3.3.1). An adjusted p-value of <= 0.01 was used as the significance threshold for the identification of differentially expressed genes.
Prior to generation of genome-wide bigWig files STAR genome BAM files were filtered using the BamTools filter command with parameters "-mapQuality 255 -isMapped true -isMateMapped true -isPaired true -isProperPair true -isPrimaryAlignment true" (Barnett et al., 2011).
Strand-specific bedGraph files were generated from filtered STAR genome BAM files using the genomeCoverageBed command from BEDTools (Quinlan & Hall, 2010) with parameters "-bg -split -scale <NORM_FACTOR> -strand <STRAND> -du". Normalisation factors generated by DESeq2 with respect to the transcriptome were used to scale the tracks. Strand was set to "-" and "+" for sense and antisense coverage, respectively. For antisense bedGraph files, negative coverage values were obtained with the command sed 's/[^\t]*/-&/4'. bedGraph files were converted to bigWig using the wigToBigWig binary available from the UCSC with the "-clip" parameter (Kent et al., 2010).
Genome_build: Ensembl R64-1-1 release 90
Supplementary_files_format_and_content: Tab-delimited text file containing TPM values generated by RSEM where gene names are rows and columns represent all RNA-Seq samples generated in the study.
Genome-wide bigWig files for both sense and antisense coverage.
|
| |
|
| Submission date |
Dec 07, 2017 |
| Last update date |
Sep 11, 2019 |
| 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 |
GPL17342 |
| Series (2) |
| GSE107813 |
Identification of non-coding transcripts regulated by the transcription factor Rap1 by RNA-Seq analysis |
| GSE110004 |
Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor |
|
| Relations |
| BioSample |
SAMN08143833 |
| SRA |
SRX3453470 |
