|
| Status |
Public on May 11, 2016 |
| Title |
Dhh1KO mRNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Whole Cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Dhh1KO (BY4741)
plasmid(s): None
|
| Treatment protocol |
Cells were harvested by vacuum filtration and flash frozen. Profiling samples were homogenized and lysed with lysis buffer using a freezer mill. Total RNA for mRNA-Seq was extraced from whole cells.
|
| Growth protocol |
Cells were grown to an OD of 0.4 in YPD media (or in dropout media in reporter/overexpression conditions as a plasmid selection strategy)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For ribosome footprint profiling, lysates were clarified and monosomes were isolated by sucrose gradient centrifugation after RNase treatment. RNA was extracted using SDS/hot phenol/chloroform. For mRNA-Seq, total RNA was extracted from frozen cells by using SDS/hot phenol/chloroform.
For ribosome footprint profiling, fragments ranging from (15/25)-34 nt were gel purified and rRNA was depleted from this pool through Ribo-Zero Gold (Yeast) treatment. Upon dephosphorylation of sample RNA, IDT miRNA Cloning Linker 1 was ligated and this product was once again gel purified. This was followed by reverse transcription as well as circularization of cDNAs. PCR amplification of this library was then sent for sequencing. For mRNA-Seq, rRNA was depleted through Ribo-Zero Gold (Yeast) treatment and the remaining RNA was randomly fragmented with light base treatment. Fragments of 20-40 nt were prepared using the above protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
rRNA Depleted Total mRNA
|
| Data processing |
Base calling was performed at Johns Hopkins or UC Riverside sequencing core facilities with CASAVA 1.8
Adapter removed with CutAdapt v.1.7.1
Low quality reads removed (Code used available at http://www.github.com/adityaradhakrishnan)
Reads mapping to ncRNA were removed (mapped with Bowtie 1.1.2 using parameters "-Sv 3 -p 4 --best --un Unmapped-*.fastq")
Reads were mapped to yeast genome (mapped with Bowtie 1.1.2 using parameters "-Sm 1 -p 4 --best --strata"
Coverage across genome computed as both counts and reads per million using 3' end mapping (Code used available at http://www.github.com/adityaradhakrishnan)
Genome_build: SacCer3 R64-1-1
Supplementary_files_format_and_content: Tab-separated text file of reads per kilobase million (RPKM) and counts for all genes under all conditions as well as fixed-step WIG density files for number of mapped counts at every nucleotide in each dataset
|
| |
|
| Submission date |
May 10, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Aditya Radhakrishnan |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Rachel Green
|
| Street address |
725 N. Wolfe St., PCTB 704
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL13821 |
| Series (1) |
| GSE81269 |
Ribosome profiling study of Dhh1p overexpression and the dhh1 knockout strain using monosome-protected footprints |
|
| Relations |
| BioSample |
SAMN04966288 |
| SRA |
SRX1753775 |
