|
| Status |
Public on May 23, 2019 |
| Title |
tif32-H725P (36 C)_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Whole cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: tif32-H725P (36 C)
|
| 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. Ribosome profiling circularized cDNA was subject to ribosomal RNA subtraction using custom biotinylated oligos in some cases. In others, Ribo-Zero was used. RNA-Seq samples were prepared with the Illumina TruSeq Stranded ribo-zero kit.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
DY171F
tif32-H725P (36 C)
Ribosome-protected RNA 25-34 nt
Biological replicate of DY170F.
Wig files composed of combination of two biological replicates (DY170F and DY171F):
processed data file: DY170F171F_plus.wig
processed data file: DY170F171F_minus.wig
|
| Data processing |
Library strategy: Ribo-seq
Base calling and demultiplexing were performed at the NHLBI DNA Sequencing and Genomics Core at the NIH Intramural program, Bethesda, MD.
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 RNA genes and those not matching were retained. PCR duplicates were removed using UMIs added during linker ligation. UMIs were then trimmed.
Reads were then aligned to the genome and those not matching to a custom library of splice junctions by using Bowtie (Langmead et al., 2009). For footprints, reads that did not match in these steps were trimmed of consecutive 3' As and realigned. The aligned reads from all alignment rounds 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.
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: R64-1-1
Supplementary_files_format_and_content: All files are fixedStep wig files (text format, each line representing one position in the genome).
|
| |
|
| Submission date |
May 02, 2019 |
| Last update date |
Jun 21, 2019 |
| Contact name |
Nicholas R Guydosh |
| E-mail(s) |
[email protected]
|
| 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 |
GPL23014 |
| Series (1) |
| GSE124204 |
Hcr1/eIF3j is a 60S ribosomal subunit recycling accessory factor in vivo |
|
| Relations |
| BioSample |
SAMN11567338 |
| SRA |
SRX5783483 |
