|
| Status |
Public on Mar 18, 2022 |
| Title |
YhgF minus crosslink biorep 3 |
| Sample type |
SRA |
| |
|
| Source name |
E. coli cultured in M9 minimal media
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: EHS-2291
condition: minus crosslink
tagged protein: 3x-FLAG tagged YhgF
protocol: CLIP-seq
|
| Treatment protocol |
Half of each culture was crosslinked by applying UV-light the other half was not treated.
|
| Growth protocol |
The strain EHS-2291 carrying a 3x-FLAG tagged YhgF was grown in M9 minimal media supplemented with 0.2 % glycerol and 0.1 % casamino acids until an OD600 of ~1.7. The experiment was done using biological independent tripicates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The YhgF protein was purified using anti-FLAG beads and bound RNA was partly digested and labeled with P32. The samples were separated on polyacrylamide gels andtransferred to nitrocellulose membranes. The RNA was prepared from the membrane by cutting out the pieces cotaining protein. Co-purifying RNA was identified by deep sequencing
Libraries were prepared using the NEBNext multiplex small RNA library prep set for Illumina (New England Biolabs) according to the manufacturer’s directions, except that 20 cycles of PCR was done after the reverse transcription. MinElute PCR purification kit (Qiagen) was used to purify and up-concentrate PCR samples. The amplified libraries were size separated on a 6 % polyacrylamide gel, stained with SYBR gold (Thermo Fisher) and fragments in the size of 140-250 bp excised from the gel. The DNA was purified using the elution buffer supplied with the kit and overnight incubation at 16 °C while shaking at 1200 rpm. To remove gel fragments from the samples, they were centrifuged through a Costar Spin-X column (0.45 µl cellulose membrane, Corning) and ethanol precipitated using linear acrylamide (from kit) as coprecipitant. To amplify the DNA library, another nine rounds of PCR was done followed again by purification and up-concentration using MinElute PCR purification kit
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
All rRNA operons +/- 50 nt except rrnH were masked from genome sequence prior to mapping.
Adapter removal and read pair merging performed with SeqPrep (https://github.com/jstjohn/SeqPrep)
Read mapping performed with bowtie 1.2.2 allowing for 1 mismatch.
Mapped reads were converted to strand separated bedgraph files with samtools and bedtools genomecov.
Bedgraph files converted to bigWig file format (www.encodeproject.org/software/bedgraphtobigwig/)
Assembly: NC_000913.3
Supplementary files format and content: Strand separated bigWig files for viewing mapping coverage in genome browsers.
|
| |
|
| Submission date |
Mar 18, 2022 |
| Last update date |
Mar 20, 2022 |
| Contact name |
Erik Holmqvist |
| E-mail(s) |
erik.holmqvist@icm.uu.se
|
| Organization name |
Uppsala University
|
| Street address |
Husargatan 3, Box 596, Uppsala University, Dept. of Cell and Molecular Biology
|
| City |
Uppsala |
| State/province |
Uppland |
| ZIP/Postal code |
75124 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL21117 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN26795525 |
| SRA |
SRX14503883 |
