|
| Status |
Public on Dec 20, 2018 |
| Title |
Rap1-AID degron induction 2 hours 5' seq replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
FW3877 (MATa leu2Δ0 met15Δ0 ura3Δ0 rap1::RAP1-3V5-IAA7::KANMX6 his3::pGPD1-osTIR::HIS3) BY4741 derivative
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: FW3877
tissue: yeast strains in small batch culture
treatment: 500 μM auxin 3-IAA (2h)
|
| 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.
|
| 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 |
polyA 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.
To obtain libraries representing the 5’ ends of polyadenylated and capped transcripts, approximately 5 µg of poly(A)+-RNA together with in-vitro spike ins were first subjected to zinc-mediated fragmentation (Ambion) at 70 °C. The reaction was cleaned up using Qiagen min-elute columns to isolate RNA fragments with mode length of ~200 nt. These fragments were then incubated with shrimp alkaline phosphatase at 37 °C to remove the 5’ phosphate groups of non-capped fragments followed by acid-phenol/chloroform extraction and ethanol precipitation. With the exception of a non-decapping control sample, dephosphorylated fragments were next treated with Cap-Clip™ acid pyrophosphatase (CellScript) to remove the 5’-terminal caps from fragments representing the bona fide 5’ ends of transcripts. After another round of acid-phenol/chloroform extraction and ethanol precipitation, all samples were treated with T4 RNA ligase 1, high concentration (NEB) to introduce a custom adapter sequence to the 5’ un-capped ends of fragments. Excess adapters were removed via a column clean up step. First strand cDNA synthesis was performed using Superscript IV (ThermoFisher) and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMix (KAPA Biosystems). Double-stranded cDNA was quantified by Qubit (Life Technologies) and used as input for library preparation using a KAPA Hyper Prep kit (KAPA Biosystems) and KAPA Single-indexed adapters (KAPA Biosystems). Libraries quantified by Qubit and were sequenced on the Illumina HiSeq 4000 platform with 75-base single-end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
5Prime-Seq
|
| Data processing |
Adapter trimming was performed with cutadapt (version 1.9.1) (Martin M, 2011) with parameters “--minimum-length=20 --quality-cutoff=20 -a AGATCGGAAGAGC”.
The primer sequence specific to the protocol was removed by rerunning cutadapt with the parameters “--minimum-length=20 --quality-cutoff=20 -g GCACTCTGAGCAATACC”, and only the reads containing the primer sequence were used for further analysis.
BWA (version 0.5.9-r16) (Li and Durbin, 2009) using default parameters was used to perform the read mapping to the S. cerevisiae genome (assembly R64-1-1, release 90; Kersey et al., 2016).
Uniquely mapped alignments corresponding to the sense and antisense strands were obtained using SAMtools view (version 1.3.1) (Li et al., 2009) with the flags “-q 1 -F 20” and “-q 1 -f 16”, respectively.
BedGraph coverage tracks representing the 5-prime signal per million mapped reads were generated using BEDTools genomeCoverageBed (version 2.26.0) (Quinlan and Hall, 2010) with the parameters “-bg -5 -scale <SCALE_FACTOR>”. 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: Genome-wide bigWig files for both sense and antisense coverage. See data processing step for more details.
|
| |
|
| Submission date |
Feb 01, 2018 |
| 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 |
GPL21656 |
| Series (2) |
| GSE110000 |
TSS identification of Rap1-regulated transcripts by 5' end RNA sequencing |
| GSE110004 |
Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor |
|
| Relations |
| BioSample |
SAMN08451827 |
