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| Status |
Public on Feb 03, 2023 |
| Title |
40_dSLT2_2CR_120min |
| Sample type |
SRA |
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| Source name |
haploid cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: delta-slt2
congo red: 2 ug/uL
timepoint: 2 hr
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| Growth protocol |
For RNAseq experiments (1-50), cells were cultured to 0.125 OD600 and either harvested directly or treated with Congo Red and harvested 1, 2, or 4 hours later. Approximately 5 OD600 units were collected by centrifugation and frozen at -80°C. For CRAC experiments (51-54), cells were cultured to 0.4 OD600 in 700 mL SD -TRP medium containing 2% glucose. 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNAseq experiments (1-50), cells were lysed with zirconia beads and vortexing, and RNA was extracted using hot phenol-chloroform. The CRAC (samples 51-54) protocol is based on Tuck and Tollervey, 2013. Modifications are described in Bresson et al., 2020.
RNAseq libraries were prepared from 500ng of each total-RNA sample using the NEBNEXT Ultra II Directional RNA Library Prep kit (NEB #7760) and the Poly-A mRNA magnetic isolation module (NEB #E7490) according to the manufacturer's protocol. The CRAC (samples 51-54) protocol is based on Tuck and Tollervey, 2013. Modifications are described in Bresson et al., 2020.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RNAseq
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| Data processing |
Alignment. Command: STAR --readFilesIn file1.fastq file2.fastq --genomeDir index_file
samtools view -bS file.sam
samtools sort file.bam
samtools index -b file.bam
Coverage map (+strand). Command: bamCoverage -b file.bam --binSize 1 --filterRNAstrand forward --normalizeUsing CPM
Coverage map (-strand). Command: bamCoverage -b file.bam --binSize 1 --filterRNAstrand reverse --normalizeUsing CPM
Read counting. Command: featureCounts file.bam -a annotation.gtf --largestOverlap --extraAttributes gene_name -s 2 -T 50 -p -B
Assembly: Saccharomyces cerevisiae EF4.74 with the introns artificially removed to improve mapping. The genome and associated annotation file are included as part of this submission.
Supplementary files format and content: The .txt files (generated using featureCounts) contain information on the number of reads mapping to each transcript. The bigwig files provide coverage maps across the yeast genome.
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 14 -at RIGHT -u 2 -q TAIL -qf i1.8
Collapsing. Command: pyFastqDuplicateRemover.py -f file.fastq
Removal of low-complexity reads. Command: bbduk.sh in=file.fasta entropy=0.65 entropywindow=10 entropyk=6
Alignment. Command: novoalign -f file.fasta -r Random -d genome.novoindex
Further collapsing of reads aligning to the same location and with identical random barcodes.
Coverage maps. Command: pyGTF2bedGraph.py --gtf annotation_file.gtf -c chromosome_lengths.txt -t reads
Read counting. Command: pyReadCounters.py -f file.bam --file_type=novo --sense --gtf=annotation_file.gtf
Assembly: 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 provide coverage maps across the yeast genome.
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| Submission date |
Aug 04, 2022 |
| Last update date |
Feb 04, 2023 |
| Contact name |
Stefan Bresson |
| E-mail(s) |
stefan.bresson@gmail.com
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| Organization name |
University of Edinburgh
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| Department |
Wellcome Trust Centre for Cell Biology
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| Lab |
Tollervey Lab
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| Street address |
Max Born Crescent, Swann 5.1
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| City |
Edinburgh |
| State/province |
Scotland |
| ZIP/Postal code |
EH9 3BF |
| Country |
United Kingdom |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE210558 |
Nab6 and Mrn1 regulate the expression of cell wall mRNAs in budding yeast |
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| Relations |
| BioSample |
SAMN30154409 |
| SRA |
SRX16849463 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6432507_40_dSLT2_2CR_120min_hittable.txt.gz |
140.8 Kb |
(ftp)(http) |
TXT |
| GSM6432507_40_dSLT2_2CR_120min_minus_strand.bw |
20.1 Mb |
(ftp)(http) |
BW |
| GSM6432507_40_dSLT2_2CR_120min_plus_strand.bw |
20.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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