|
| Status |
Public on Jul 23, 2014 |
| Title |
Sc_BY4741-Sub2 |
| Sample type |
SRA |
| |
|
| Source name |
PAR-CLIP of Sub2-TAP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
tagged protein: Sub2-TAP
growth medium: Minimal complete medium with 4tU
|
| Treatment protocol |
4tU-labeled yeast cells were UV-irradiated with an energy dose of 12 J/cm2 at 365 nm.
|
| Growth protocol |
Yeast cells expressing the TAP-tagged protein were grown at 30 °C from OD600 ∼0.1 to OD600 ∼0.5 in CSM minimal medium (Formedium) supplemented with 10 mg/l uracil, 100 µM 4-thiouracil (4tU) and 2 % glucose. After reaching OD600∼0.5, another 900 µM 4tU were added and cells were grown further for 4 h (OD600∼1.3-1.6).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed by bead beating and the lysate was sonicated. Immunoprecipitation was performed on a rotating wheel for 2 h at 4 °C with rabbit IgG-conjugated Protein G magnetic Dynabeads (Invitrogen). Crosslinked RNA was partially digested with RNase T1 and prepared for cDNA library preparation.
Data acquisition were performed as described by Schulz and Schwalb et al. (Cell, 2013) with minor modifications. Following adapter ligation, RNA was recovered by Proteinase K digestion for 2 h at 55 °C, and subsequent acidic phenol/chloroform extraction. Reverse transcription was done for 1 hr at 44 °C using SuperScript III RTase (Invitrogen). PCR amplification was performed using the NEXTflex barcode primer kit (Bioo Scientific).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Protocol: PAR-CLIP
|
| Data processing |
Adapter sequences are first trimmed from the raw sequencing files. The quality filter then discards all reads containing unidentified nucleotides (N), Phreds scores below 30, reads shorter than 15nt, or reads that are flagged by Illumina’s internal chastity filter. Quality-trimmed reads are aligned to the S. cerevisiae genome (sacCer3, version 64.1.1) using the short read aligner Bowtie (version 0.12.7) with a maximum of one mismatch and taking unique matches only (options: -q -p 4 -S -sam -nohead -v 1 -e 70 -l 28 -y -a -m 1 -best -strata -phred33 -quals)
Genome_build: S. cerevisiae genome (sacCer3, version 64.1.1)
Supplementary_files_format_and_content: tab delimited text files containing genomic locations, p-values and occupancies
|
| |
|
| Submission date |
Jul 22, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Carlo Bäjen |
| E-mail(s) |
carlo.baejen@mpibpc.mpg.de
|
| Organization name |
Max Planck Institute for Biophysical Chemistry
|
| Department |
Molecular Biology
|
| Lab |
Prof. Patrick Cramer
|
| Street address |
Am Fassberg 11
|
| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13272 |
| Series (1) |
| GSE59676 |
Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition |
|
| Relations |
| BioSample |
SAMN02929663 |
| SRA |
SRX659669 |
