 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 06, 2023 |
| Title |
3KO 1h post sporulation induction, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
strain 168
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: strain 168
genotype: 3KO
library type: RIBO-seq
treatment: bacterial cells
|
| Growth protocol |
The B. subtilis 168 strain served as the wild type, and other strains were derived from it. The strains were cultured overnight in LB medium at 30 °C with shaking, then diluted to OD600=0.1 in CH medium. They were grown until OD600 reached 0.5 – 0.6 at 37 °C with shaking. Sporulation was induced by changing the medium to sporulation medium, following the method by Sterlini and Mandelstam (1968), with the modification that cells were harvested by filtration. Filters were then transferred into the culture flasks, and the sporulation medium was supplemented with 3% v/v of the culture in the CH medium at OD600=0.5-0.6 to promote sporulation.
|
| Extracted molecule |
other |
| Extraction protocol |
RNA was isolated from sporulating WT and 3KO strains. Cultures were harvested hourly for seven hours into sporulation, beginning at T0 (prior to sporulation induction), resulting in a total of eight timepoints. Before harvesting, cultures were treated with 0.3 mM chloramphenicol. Cells were collected by filtration and flash frozen in liquid nitrogen. Purification of mRNA, ribosomal footprint isolation and library preparation was performed as described earlier. Briefly, frozen pellets were ground using mortar and pestle with the addition of aluminum oxide and lysis buffer. The extracted total RNA was used for purification of mRNA and ribosomal footprint isolation. rRNA depletion was performed to yield mRNA which was then fragmented by alkaline hydrolysis. Ribosomal footprint isolation was performed by MNase digestion of the total RNA followed by size selection using polyacrylamide gel electrophoresis.
The obtained mRNA fragments and ribosomal footprints were end-repaired (dephosphorylation of 3'ends followed by phosphorylation of 5' ends) and used for preparation of cDNA sequencing libraries with NEBNext Multiplex Small RNA Library Prep Set for Illumina, according to the manufacturer's guidelines. Sequencing was performed on an Illumina NextSeq500 by Genomed S.A. (Warsaw, Poland) and Genome Facility at CeNT (Warsaw, Poland); single-end 50bp reads were sequenced.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
3KO1r_2
countsRAW_CDSandUTR_Ribo.xlsx
countsRAW_CDS_Ribo.xlsx
countsTPM_CDSandUTR_Ribo.xlsx
countsTPM_CDS_Ribo.xlsx
DESeq2_betweenConditions_CDS_batchCorrected_Ribo.xlsx
|
| Data processing |
FastQC reports were utilized to assess raw data quality.
Following this step, the removal of adapters and low-quality sequences was performed using TrimGalore! software
The Bowtie tool was employed to eliminate sequences corresponding to tRNA and rRNA using genome reference data.
For RIBO-seq data, filtering was applied, retaining only sequences between 15-32 nucleotides in length.
After preprocessing, the FastQC tool was used for quality control.
Length distribution plots were created to analyze the read length distribution of the RIBO-seq data.
The clean and trimmed data was mapped to the genome using STAR, with a tolerance of up to 4% mismatch in read length and exclusion of splicing. The resulting BAM files were sorted and indexed using the sambamba software. Unique mapped reads were counted using featureCounts, employing gene and UTR annotations from the BSGatlas database, with a minimum overlap of 4 nucleotides.
Subsequently, the normalization of read counts was carried out using the Transcripts Per Million (TPM) method for CDS, UTR, and combined data.
Differential gene expression analysis was carried out on RNA-seq and RIBO-seq data using the DEBrowser tool and the DESeq2 library.
Assembly: NC_000964.3
Supplementary files format and content: XLSX files include raw counts for each sample (CDS and UTRs)
Supplementary files format and content: XLSX files include raw counts for each sample (CDS only)
Supplementary files format and content: XLSX files include TPM values for each sample (CDS and UTRs)
Supplementary files format and content: XLSX files include TPM values for each sample (CDS only)
Supplementary files format and content: XLSX file includes differential expression results between 3KO and WT for each time-point
Library strategy: RIBO-seq
|
| |
|
| Submission date |
Dec 05, 2023 |
| Last update date |
Dec 06, 2023 |
| Contact name |
Agata L. Starosta |
| E-mail(s) |
agata.starosta@ibb.waw.pl
|
| Organization name |
Institute of Biochemistry and Biophysics Polish Academy of Sciences
|
| Lab |
Laboratory of Translatomics
|
| Street address |
Pawińskiego 5a
|
| City |
Warsaw |
| ZIP/Postal code |
02-106 |
| Country |
Poland |
| |
|
| Platform ID |
GPL24109 |
| Series (2) |
| GSE249448 |
Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation [Ribo-seq] |
| GSE249450 |
Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation |
|
| Relations |
| BioSample |
SAMN38687203 |
| SRA |
SRX22793009 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
