 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 28, 2019 |
| Title |
Csl4_1_PARCLIP |
| Sample type |
SRA |
| |
|
| Source name |
Yeast cell extract
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain background: BY4741
degradation factor: Csl4
|
| Treatment protocol |
1 mM 4tU for 4 h.
|
| Growth protocol |
4tU-labeled yeast cells (BY4741) expressing C-terminally TAP-tagged protein were UV-irradiated with an energy dose of 10-12 J/cm2 at 365 nm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were lysed by bead beating and IP was performed with rabbit IgG-conjugated protein G magnetic Dynabeads (Invitrogen). The cross-linked RNA was partially digested with RNase T1 (Thermo Scientific) and the IP was controlled by performing a western blot analysis of a part of the sample using the Peroxidase Anti-Peroxidase (PAP; Sigma) antibody. Following adapter ligation, RNA was recovered by digestion with proteinase K (New England Biolabs) and a subsequent acidic phenol/chloroform extraction.
Reverse transcription was performed with SuperScript III RTase (Invitrogen) and was followed by PCR amplification using the NEXTflex barcode primer kit (Bio Scientific). The generated cDNA was purified, size selected, and quantified using TapeStation (Agilent Technologies). The resulting samples were then PCR-amplified and sequenced on an Illumina HiSeq 1500, HiSeq2500 or NextSeq550 system.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Library strategy: PAR-CLIP
Adapter sequences are trimmed and a quality filter discards reads containing unidentified nucleotides (N), Phred scores below 20, or reads shorter than 20
High-quality reads are aligned to S. cerevisiae genome (sacCer3, version 64.1.1) using Bowtie (v1.2). Reads are mapped uniquely with a maximum of one tolerated mismatch
Mockinbird is used for statistical evaluation of binding sites. A p-value threshold of 0.005 was set as well as the minimum coverage of 2 per site.
Genome_build: S. cerevisiae genome (sacCer3, version 64.1.1)
Supplementary_files_format_and_content: tab delimited text files containing verified cross-link sites (p-value<0.005), their genomic locations, p-values and occupancies
|
| |
|
| Submission date |
Mar 14, 2019 |
| Last update date |
May 28, 2019 |
| Contact name |
Katharina Bettina Hofmann |
| Organization name |
Max Planck Institute for Biophysical Chemistry
|
| Department |
Molecular Biology
|
| Street address |
Am Fassberg 11
|
| City |
Goettingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL26302 |
| Series (1) |
| GSE128312 |
Transcriptome maps of general eukaryotic RNA degradation factors |
|
| Relations |
| BioSample |
SAMN11127523 |
| SRA |
SRX5522644 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3671248_Csl4.table.txt.gz |
3.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
