|
| Status |
Public on Mar 25, 2024 |
| Title |
mutL*//polC* strain, without IPTG, replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
planktonic exponential phase (0D600 ~0.4)
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
cell type: planktonic exponential phase (0D600 ~0.4)
genotype: mutL*//polC*
treatment: no IPTG
|
| Treatment protocol |
Samples were distributed in tubes containing 15mL of frozen killing buffer (20 mM Tris-HCl, ph 7.5, 5 mM MgCl2, 20 mM NaN3).
|
| Growth protocol |
A single clone was cultured overnight in 10 mL in LB at 37°C. This preculture was diluted to OD600 = 0.01 in 2 mL of LB and grown at 37°C. When the culture reached OD600 = 0.4, it was diluted by 40 to OD600 = 0.01 in 5 mL.
This cycle of growth and dilution to OD600 = 0.01 was repeated twice in growing volumes (20 mL then 150 mL culture). In conditions with IPTG, 100µM IPTG was added in the 20 mL culture.
When the 150 mL culture reached OD600 = 0.4, 3 x 30 mL of culture were sampled.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by acid-phenol extraction protocol consisting of two extractions with an equal volume of acid phenol/chloroform/isoamyl alcohol (25:24:1, [pH 4.5]) followed by one extraction with chloroform/isoamyl alcohol (24:1).
After adding 1/10 volume of 3 M sodium acetate (pH 5.2), RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in 100 μl of RNase free water.
For transcriptome analysis, 35 μg RNA were DNase-treated using the RNase-Free DNase Set (Qiagen) and purified using the RNA Clean-Up and Concentration Micro Kit (Norgen).
Library construction -including ribosomal RNA depletion- and sequencing was performed by Fasteris (Plan-les-Ouates Switzerland).
RNA depletion was performed with the Lexogen RiboCop META kit, library preparation used the Illumina TruSeq stranded mRNA kit, and the sequencing was paired-end (2 × 50 bp on Illumina NovaSeq 6000).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
mutLpolC_no_IPTG_rep_1
|
| Data processing |
Base calling was performed by Fasteris using NovaSeq Control Software 1.7.5, RTA v3.4.4, and bcl2fastq2.20 v2.20.0.422.
Reads were trimmed using sickle 1.33 with options-t sanger -x -q 20 -l 20.
Alignment to the reference sequence of the B. subtilis 168 genome (Genbank AL009126.3) used the BWA-MEM alignment tool version 0.7.17
Stranded read counts per gene annotated in AL009126.3 were obtained with htseq-count 2.0.1 (options “-s reverse -m union --nonunique all")
Fragment counts normalised per kilobase of feature length per million mapped fragments (FPKM) were computed with DESeq2 v1.38.3 based on robust estimation of library size.
Assembly: AL009126.3
Supplementary files format and content: tab-delimited text file
Supplementary files format and content: First six columns : location of the gene, locus tag, and gene name (as annotated in AL009126.3)
Supplementary files format and content: Next 12 columns : raw counts for each sample
Supplementary files format and content: Last 12 columns : normalized expression levels (FPKM) for each sample
|
| |
|
| Submission date |
Aug 01, 2023 |
| Last update date |
Mar 25, 2024 |
| Contact name |
Ira Tanneur |
| E-mail(s) |
irene.tanneur@inrae.fr
|
| Organization name |
INRAE
|
| Department |
MaIAGE
|
| Street address |
INRAE - Unité MaIAGE Bât 210 et 233 Domaine de Vilvert
|
| City |
JOUY-EN-JOSAS |
| ZIP/Postal code |
78352 |
| Country |
France |
| |
|
| Platform ID |
GPL30358 |
| Series (1) |
| GSE239804 |
Exploring the Respective Contributions of DNA Polymerase Proofreading and Mismatch Repair in the Shaping of Spontaneous Mutation Rates. |
|
| Relations |
| BioSample |
SAMN36795398 |
| SRA |
SRX21218583 |
