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| Status |
Public on May 21, 2021 |
| Title |
Ycg1Degron2 Untreated TT-seq replicate 3 |
| Sample type |
SRA |
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| Source name |
yeast strains in small batch culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: W303-1A, Mat a, (K.lactis URA3)pMET3-YCG1-3PK-miniAID::KANMX6, ADE2::pADH1-OsTIR1-9Myc
treatment: Untreated
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| Treatment protocol |
The control and Ycg1Degron2 were treated identically. To deplete condensin, cells were grown overnight in YNB + CSM -methioninie medium, synchronised from an OD600 of 0.2 in G1 for 2 hours then released into fresh YNB + CSM -methionine medium containing nocodozole for 1.5 hours to achieve a metaphase arrest. Methionine was added at a final concentration of 133μg/ml to shut off the MET3 promoter and indole-acetic acid (IAA, auxin) at a final concentration of 88μg/ml to degrade Ycg1 for 30 minutes. 4-TU was added to the samples at a final concentration of 5mM for 5 minutes before collection from the control and degron strains with and without methionine and auxin treatment. A sample from each strain which had undergone auxin and methionine treatment was then heat shocked for 15minutes at 37°C for 15 minutes, followed by a 5 minute 4-TU pulse before collection
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| Growth protocol |
Cells were grown overnight in YNB supplemented with CSM -methionine. Cells were grown at 25°C, shaking at 200 RPM in incubators.
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| 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 cleaned up through column purification through the RNA Cleanup protocol within the RNeasy Mini Kit including DNase digestion (Qiagen). To measure global transcription levels a 4-TU pulsed S. pombe Total RNA was used as a spike-in for all samples, 90μg of sample Total RNA was combined with 10μg of S. pombe Total RNA. Nascent RNA from the samples containing also S. pombe spike-in RNA was isolated using TTchem-seq, this involved biotinylation of chemically fragmented Total RNA, the biotiylated nascent RNA was isolated from the fragmented Total RNA using a bead-based streptavidin pull down. 20 ng of nascent RNA was used to make libraries with the KAPA RNA HyperPrep Kit (Roche). The library was made following the manufacturer’s instructions, with adjustments to ensure small transcripts from the fragmentation were not lost. The ratio of KAPA pure beads to adapter-ligated cDNA was 0.95x in the 1st post-ligation clean up, and 1x in the 2nd post-ligation clean up. Libary quailty was assessed using a Biolanalyzer 2100 (Agilent) and nascent RNA sequencing was carried out on the Illumina HiSeq 4000 platform and typically generated approximately 19 million 76 bp strand- specific single-end reads.
library strategy: TT-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Adapter trimming was performed with cutadapt (version 1.9.1) (Martin, 2011) with parameters “--minimum-length=20 --quality-cutoff=20 -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, uniquely mapped, and had fewer than 3 mismatches using BamTools (version 2.4.0; (Barnett et al, 2011)).
Alignments corresponding to the sense and antisense strands were obtained using SAMtools view (version 1.3.1) (Li et al, 2009) by using the flags “-f 16” and “-F 20”, respectively.
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 -s 2”.
BedGraph tracks were created using the BEDTools genomeCoverageBed (version 2.26.0) (Quinlan & Hall, 2010) by normalising the genome-wide coverage relative to DESeq2 size factors generated with respect to the S. pombe transcriptome. The parameters used were “-bg -pc -strand <STRAND> -scale <SCALE_FACTOR>”. 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 raw counts generated by featureCounts where gene names are rows and columns represent all samples generated in the study.
Supplementary_files_format_and_content: Genome-wide bigWig files for both sense and antisense coverage. See data processing step for more details.
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| Submission date |
Nov 16, 2020 |
| Last update date |
May 21, 2021 |
| Contact name |
Lucy Lancaster |
| E-mail(s) |
lucy.lancaster@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| Lab |
Chromosome Segregation Laboratory
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| Street address |
1 Midland Rd
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| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platform ID |
GPL21656 |
| Series (2) |
| GSE161581 |
A role for condensin in mediating transcriptional adaptation to environmental stimuli. [TT-Seq] |
| GSE161582 |
A role for condensin in mediating transcriptional adaptation to environmental stimuli. |
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| Relations |
| BioSample |
SAMN16811638 |
| SRA |
SRX9516445 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4910518_34_CT_R3.antisense.bigWig |
9.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM4910518_34_CT_R3.sense.bigWig |
9.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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