|
| Status |
Public on Mar 20, 2020 |
| Title |
02_cbf1_CLIP_XL |
| Sample type |
SRA |
| |
|
| Source name |
RNA fragments bound to Cbf1
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: TIGR4
growth phase: Mid-exponential (OD 0.5)
genotype: cbf1-3xFLAG
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were either irradiated (XL) by UV light or not (ctrl). Following immunoprecipitation and trimming of the RNA bound to Cbf1, the RNA was radioactively labeled and the RNA-Cbf1 complexes were transferred to a membrane by western blotting. The RNA was then extracted from the membrane by digestion of the protein.
After ligation of 3' and 5' adapters, cDNA preparation was performed and the resulting cDNA was amplified by PCR. Fragments between 140-250 bp were size-selected from a gel and again amplified by PCR to obtain the final library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
crosslinked sample
|
| Data processing |
Quality and adapter trimming was performed using Cutadapt 1.17 (cutoff Phred score of 20 in NextSeq mode).
Reads >11 nt were aligned using READemption 0.4.5 and segemehl 0.2.0 (accuracy cutoff of 80%).
Normalization was performed by filtering positions with read counts <6 standard deviations from 0. The distribution ratios between the paired XL and ctrl samples presented a clear bimodal distribution representing the background and crosslink-enriched positions, which were separated by k-means clustering. Size factors were calculated from the background positions present across all replicates against a geometric mean pseudo-reference, as introduced in DESeq.
Peak calling was performed using PEAKachu 0.1.0 setting the maximum fragment size to 50 and using the previously calculated size factors.
Genome_build: NC_003028.3
Supplementary_files_format_and_content: wig files contain normalized coverage information from the CLIP-seq experiment
|
| |
|
| Submission date |
Feb 20, 2020 |
| Last update date |
Mar 21, 2020 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
|
| Organization name |
ZB MED - Information Centre for Life Sciences
|
| Department |
Information Services
|
| Lab |
Förstner Lab
|
| Street address |
Gleueler Str. 60
|
| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23797 |
| Series (2) |
| GSE145604 |
Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control |
| GSE145605 |
Exonuclease-mediated sRNA activation in competence regulation in a Gram-positive bacterium |
|
| Relations |
| BioSample |
SAMN14144296 |
| SRA |
SRX7756606 |
