|
| Status |
Public on Dec 06, 2017 |
| Title |
untagged control 3 |
| Sample type |
SRA |
| |
|
| Source name |
bacterial cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: K12 MG1655
genotype: wild type
|
| Treatment protocol |
Samples were treated with 400mJ of UV light at 254nM. They were then quickly cooled, washed, and flash frozen in liquid nitrogen.
|
| Growth protocol |
Strains were grown in LB media to mid-exponential phase (OD600 = 0.5)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were thawed, lysed with sonication over ice, and cleared with centrifugation. CsrA-RNA complexes were immunoprecipated with M2 anti-FLAG mouse antibody conjugated paramagnetic particles (Sigma) for 4 hours at 4C. Complexes were washed, 5'-end labeled with 32P, and eluted from the particles by heating in non-reducing PAGE sample buffer. Protein-RNA complexes were seperated with SDS-PAGE and transferred to nitrocellulose. RNA was extracted from the nitrocellulose by treatment with proteinase K, phenol chloroform extraction, and ethanol precipitation.
NEB small RNA library prep kit (Catalog #: E7300S)
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalls were made with Casava.
Reads were trimmed to remove adaptor sequences and reads shorter than 12 bases were discarded. Reads were aligned to the E. coli rRNA sequences with bowtie with default parameters and unaligned reads were retained
Reads were aligned to the E. coli genome with bowtie using default parameters. PCR duplicates were removed using Picard MarkDuplicates
All 3xFLAG-CsrA samples were merged into a single sam file and used as input into PIPE-CLIP to identify CLIP-seq peaks. HTSeq Count was used to count reads per peak in each individual sample bam file using the identified peaks
DESeq2 was used to identify peaks significantly enriched over the untagged control samples using the individual sample count data
Genome_build: NC_000913.3
Supplementary_files_format_and_content: csv file with peaks and sample counts
|
| |
|
| Submission date |
Aug 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Anastasia Haley Potts |
| E-mail(s) |
anastasi@ufl.edu
|
| Organization name |
University of Florida
|
| Department |
Department of Microbiology and Cell Science
|
| Lab |
Dr. Tony Romeo
|
| Street address |
1355 Museum Drive, Bldg. 981
|
| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32611 |
| Country |
USA |
| |
|
| Platform ID |
GPL15010 |
| Series (2) |
| GSE102380 |
RNA binding partners of the post-transcriptional regulator CsrA |
| GSE102386 |
The contribution of the post-transcriptional regulator CsrA to RNA stability and translation and identification of binding partners |
|
| Relations |
| BioSample |
SAMN07469492 |
| SRA |
SRX3073811 |
