|
| Status |
Public on Sep 14, 2017 |
| Title |
ski2KO dom34KO mRNA-Seq+DTT |
| Sample type |
SRA |
| |
|
| Source name |
Whole cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: ski2KO dom34KO
cell culture treatment: DTT
|
| Treatment protocol |
Cells were rapidly vacuum filtered and flash frozen. Ribosome profiling samples were lysed in a freezer mill with lysis buffer. Total RNA for mRNA-Seq was extracted from whole cells.
|
| Growth protocol |
Cells were generally grown to an OD of ~0.6 and harvested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ribosome profiling lysates were clarified, RNase treated, and run over sucrose gradients to extract the monosome peak. RNA was then extracted using SDS/hot phenol/chloroform and footprints of the specified size were retained by gel purification.
Briefly, RNAs were ligated to a universal DNA linker followed by reverse transcription. cDNAs were then circularized and PCR amplified. Illumina Ribo-Zero kits were used to remove rRNA (from total RNA for mRNA-Seq, from size-selected footprints for 51F-56F, and from linker-ligated footprints for 63F and 64F).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Base calling was performed at Johns Hopkins sequencing facilities.
Files were demultiplexed and filtered to remove low-quality reads. They were then trimmed to the start of the linker sequence. Contaminating sequences that corresponded to gel markers were eliminated.
Reads were aligned to a library of noncoding RNA genes and those not matching were retained.
Reads were then aligned to the genome and those not matching to a custom library of transcripts by using Bowtie (Langmead et al., 2009) and the parameters: -y -a -m 1 --best --strata. Reads were pooled and normalized to units of reads per million (rpm) (by dividing by the total number of million mapped reads in a sample) to generate wig files. Wig files beginning with "end3" are aligned by 3' ends, all others by 5' ends. 0 mismatches were tolerated for footprint libraries, 2 mismatches for RNA-Seq libraries.
Replicates or multiple sequencing lanes for a given sample were pooled together to create a single wig file for each condition to be used in downstream analysis.
Genome_build: PomBase ASM294 v2.22
Supplementary_files_format_and_content: All files are fixedStep wig files (text format, each line representing one position in the genome).
|
| |
|
| Submission date |
May 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas R Guydosh |
| E-mail(s) |
nicholas.guydosh@nih.gov
|
| Organization name |
National Institutes of Health
|
| Department |
NIDDK
|
| Lab |
LBG
|
| Street address |
8 Center Dr, Bldg 8, 220
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL17225 |
| Series (1) |
| GSE98934 |
Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe |
|
| Relations |
| BioSample |
SAMN07125193 |
| SRA |
SRX2825811 |
