|
| Status |
Public on Oct 17, 2013 |
| Title |
3h_MM1S_20nM_bortezomib_polyA_mRNA |
| Sample type |
SRA |
| |
|
| Source name |
MM1.S myeloma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cycloheximide: none
time point: 3h
sample type: poly(A) mRNA
|
| Treatment protocol |
0h samples untreated, all others 20 nM bortezomib treated. Ribosome footprint samples treated with 0.1 mg/mL cycloheximide for 1 min immediately prior to harvesting.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Ribosome footprints were isolated from 80S ribosomes by ultracentrifugation followed by gel-based size selection, as previously described (Ingolia et al, Cell 2011). Poly(A) mRNA was isolated using poly-d(T) coated magnetic beads, then size-selected by PAGE after alkaline fragmentation.
Libraries were constructed as previously described (Ingolia et al, 2011). Briefly, size-selected ribosome footprint and poly(A) mRNA fragments were ligated to Linker-1 oligonucleotide (IDT) by T4 RNA ligase following PNK dephosphorylation. Ligated RNA was purified after gel-based separation. Reverse transcription to cDNA was carried out using SuperScript III enzyme. cDNA fragments were purified after gel-based separation. Ribosomal RNA subtraction was performed on ribosome footprint samples using biotinylated oligonucleotides (listed in Stern-Ginossar et al, Science 2012) and streptavidin-coated magnetic beads. Samples were circularized using Circ Ligase (Epicentre) and PCR amplified using primers containing Illumina barcodes for final libraries. Up to four samples were multiplexed in a single lane for sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base-calling: CASAVA v. 1.7
Demultiplexing: Sample barcodes and linker-1 sequence were removed from reads using in-house Python script (raw data files provided after this step)
Alignment: bowtie v.0.7 with up to two mismatches allowed. Reads were first aligned versus human rRNA sequences and tRNA sequences with only unaligned sequenced carried through for analysis. Reads were then aligned versus canonical transcripts from build hg19 (UCSC) and unaligned reads were aligned against the full hg19 build.
Read assignment to transcripts: in-house C++ script. Only reads uniquely mapping to a single location in a canonical hg19 transcript were included for analysis.
Read normalization: RPKM based on total number of uniquely assigned reads from in-house C++ script and hg19 canonical transcript length (processed file provided after this step)
Genome_build: hg19
Supplementary_files_format_and_content: raw reads and RPKM values in attached Excel files indicate read assignments to each UCSC transcript identifier
|
| |
|
| Submission date |
Jul 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Arun P. Wiita |
| Organization name |
University of California, San Francisco
|
| Department |
Laboratory Medicine
|
| Street address |
185 Berry St., Suite 290
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94107 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE48785 |
Global response to chemotherapy-induced apoptosis |
|
| Relations |
| BioSample |
SAMN02231212 |
| SRA |
SRX320147 |
