|
| Status |
Public on Jan 01, 2024 |
| Title |
RIBO_B.s_BY212_Cm_64_2 |
| Sample type |
SRA |
| |
|
| Source name |
Gram-positive bacterium cell
|
| Organism |
Bacillus subtilis |
| Characteristics |
cell type: Gram-positive bacterium cell
strain: BY212
genotype: amylase production
treatment: Chloramphenicol
|
| Treatment protocol |
40 ml cultures were poured into 2 Falcon tubes (50 ml) containing each 20 ml crushed frozen PBS buffer and 1 mM (323 µg/ml) chloramphenicol.
|
| Growth protocol |
Fresh transformants of BY212 were streaked out on LB plates containing 20 μg/ml kanamycin at 37 °C. The next day cells were scraped from the plate and resuspended in 0.9% NaCl and subsequently diluted into 40 ml B3 medium containing 20 μg/ml kanamycin to an OD600 of approximately 0.25, and grown at 30 °C for 88 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The culture was cryogenically pulverized to release intracellular substances. And the ribosome bound with RNA was pelleted by ultracentrifugation over a sucrose cushion. The ribosome bound with RNA was digested with micrococcal nuclease and then was ultracentrifuged over a 10-50% sucrose gradient. 30%, 40% and 50% sucrose fractions were collected and used for RNA extraction by using phenol/chloroform/isoamyl alcohol. Ribosome protected fragments (RPFs) were isolated form extracted RNA by electrophoresis on a TBE 7M Urea PAGE gel and used for construction of cDNA libraries.
1 µl of RNA fragments were used to measure the RNA concentration using the Qubit RNA HS assay kit (Thermo Fisher). Both for the RPF and the unprotected mRNA fragments sequencing libraries were constructed using the Small RNA Library Prep Set for Illumina (NEB), following the manufacturer's protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
64 h Ribo-seq replicate 2
|
| Data processing |
After deep sequencing, the raw sequence reads of both RPFs and unprotected mRNA fragments were uploaded into the Galaxy platform (https://usegalaxy.org/).
Using the Cutadapt tool (version 4.0+galaxy0), the 3’ adapter sequences were removed, and 18 to 34 nt-long reads were selected, which on average removes approximately 15% of the total reads.
After checking that the adapter has been removed from reads by using the FastQC tool (Galaxy Version 0.73+galaxy0), the Bowtie2 tool (Galaxy Version 2.4.2+galaxy0) was used to align reads to the B. subtilis genome sequence NC_000964.3 to generate SAM files, which link reads to their genomic position.
Assembly: Bacillus.fasta
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Supplementary files format and content: bigwig file includes RPKM vaules of each sample
Library strategy: Ribo-seq
|
| |
|
| Submission date |
Dec 15, 2023 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Yaozu Han |
| E-mail(s) |
Y.han@uva.nl
|
| Organization name |
Unversity Of Amsterdam
|
| Street address |
Science Park 904
|
| City |
Amsterdam |
| ZIP/Postal code |
1098 XH Amsterdam |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL28092 |
| Series (2) |
| GSE250313 |
Ribosome profiling reveals conserved guanosine residues at ribosome pausing sites in Bacillus subtilis [Ribo-seq] |
| GSE250314 |
Ribosome profiling reveals conserved guanosine residues at ribosome pausing sites in Bacillus subtilis |
|
| Relations |
| BioSample |
SAMN37950439 |
| SRA |
SRX22366403 |
