|
| Status |
Public on Aug 14, 2020 |
| Title |
P_deltaSki2_control_RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
deltaSki2_control_RNAseq
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: deltaSki2
cell/ploidy type: haploid cells
treatment: control
|
| Growth protocol |
For CRAC experiments (07-99), cells were cultured in SC -TRP medium containing 2% glucose to 0.4 OD. For the 'control', cells were then subjected to 100 mJ of 254nm UV-crosslinking (4-6 seconds), collected by filtration, resuspended in 50 mL of ice cold PBS, centrifuged to pellet, and frozen at -80°C. For all other conditions, cells at 0.4 OD were first collected by filtration, then transferred to the appropriate medium and cultured for either 30 seconds or 16 minutes prior to UV-crosslinking. All subsequent harvesting steps were identical to the control. For RNAseq experiments (A-S), we used the same approach as for CRAC but without the crosslinking step.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The CRAC protocol is based on Tuck and Tollervey, 2013. Modifications are described in Bresson and Tollervey, 2020.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Demultiplexing. Command: pyBarcodeFilter.py -f raw_file.fastq -b barcodes_file.txt
Adapter stripping and quality control. Command: flexbar -r file.fastq -a adapters.txt -ao 4 -m 11 -at RIGHT -u 2 -q TAIL -qf i1.8
Collapsing. Command: pyFastqDuplicateRemover.py -f file.fastq (Note that this step was used only for the CRAC analysis. RNAseq files were not collapsed)
Removal of low-complexity reads. Command: bbduk.sh in=file.fasta entropy=0.5 entropywindow=10 entropyk=6
Alignment. Command: novoalign -f file.fasta -r Random -o SAM -d genome.novoindex
samtools view -bS file.sam
samtools sort file.bam
Bedgraph files (+ strand). Command: bedtools genomecov -split -strand + -ibam sorted_file.bam -bg -scale variable > file_plus_strand.bedgraph
Bedgraph files (- strand). Command: bedtools genomecov -split -strand - -ibam sorted_file.bam -bg -scale variable > file_minus_strand.bedgraph
Read counting. Command: pyReadCounters.py -f file.bam --file_type=bam --sense --gtf=annotation_file.gtf
Genome_build: Saccharomyces cerevisiae EF4.74 with the introns artificially removed to improve mapping
Supplementary_files_format_and_content: The .txt files (generated using pyReadCounters.py) contain information on the number of reads mapping to each transcript. The bedgraph files show the distribution of reads across the genome. Individual replicates were merged into a single file to generate the bedgraph files.
|
| |
|
| Submission date |
Aug 13, 2020 |
| Last update date |
Aug 14, 2020 |
| Contact name |
Stefan Bresson |
| E-mail(s) |
stefan.bresson@gmail.com
|
| Organization name |
University of Edinburgh
|
| Department |
Wellcome Trust Centre for Cell Biology
|
| Lab |
Tollervey Lab
|
| Street address |
Max Born Crescent, Swann 5.1
|
| City |
Edinburgh |
| State/province |
Scotland |
| ZIP/Postal code |
EH9 3BF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE148166 |
Stress-induced translation inhibition through release of 40S scanning initiation factors |
|
| Relations |
| BioSample |
SAMN15813392 |
| SRA |
SRX8943712 |
