|
| Status |
Public on May 31, 2021 |
| Title |
WT rep3 |
| Sample type |
SRA |
| |
|
| Source name |
bacterial culture
|
| Organism |
Mycolicibacterium smegmatis |
| Characteristics |
strain: MC2 155
phase: logarithmic
genotype: WT
|
| Growth protocol |
M.smegmatis MC2 155 WT and ΔF6 strains were grown up to the logarithmic phase (OD ~ 1.0) in Middlebrook 7H9 media supplement with 0.05% Tween 80.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cultures were grown up to the logarithmic phase (OD ~ 1.0), centrifuged (4°C, 3000 rpm) and total RNA was isolated by phenol-chloroform extraction after cell disruption by Bead Beater with 0.1 mm zirconia beads (BioSpec Products, USA) as previously described (Rustad et al., 2009). After isolation, RNA was treated with Turbo DNase (Life Technologies, USA) to remove traces of genomic DNA, and purified with the RNeasy mini kit (Qiagen, Netherlands). Amounts and purity of RNA were determined spectrophotometrically; integrity of RNA was assessed in 1% agarose gel.
RNA samples were depleted of rRNA using Ribo-Zero rRNA Removal Kit (Bacteria). Sequencing libraries were generated using the resulting ribosomal transcript-depleted RNA and NEBNext Ultra II Directional RNA Library Prep Kit (NEB, USA) according to the manufacturers' protocol. Sequencing was performed in triplicates using the Illumina HiSeq2500 as the pair-ended 150 nt-long reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were mapped to M.tuberculosis H37Rv genome using bowtie2 v2.3.4.3 with parameters -p 4 --local --dovetail
Fragments counting was performed using FeatureCounts (Annotation of the genome was retrieved from Mycobrowser). For every annotated gene, the number of fragments overlapping the gene at least by one nucleotide was calculated. Only unambiguously mapped, non-chimeric fragments were used. The files "Counts_XXX.txt" represent these counts (File "Counts_all.csv" contains values for all samples).
Search for differentially expressed genes was done with DESeq2 package (using only CDS transcripts). The results are represented in file "DifferentialExpression.csv"
Genome_build: NC_008596.1
Supplementary_files_format_and_content: Counts - the numbers of overlapping reads for every annotated gene in each sample; DifferentialExpression.csv - the results of search for differentially expressed genes using DESeq2 package.
|
| |
|
| Submission date |
Apr 23, 2020 |
| Last update date |
May 31, 2021 |
| Contact name |
Artem Grigorov |
| E-mail(s) |
artgrigorov@gmail.com
|
| Organization name |
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
|
| Department |
Genomics and Postgenomic Technologies
|
| Lab |
Regulatory Transcriptomics
|
| Street address |
Ulitsa Miklukho-Maklaya, 16/10
|
| City |
Moscow |
| ZIP/Postal code |
117997 |
| Country |
Russia |
| |
|
| Platform ID |
GPL26210 |
| Series (1) |
| GSE149173 |
RNA-seq analysis of Mycobacterium Smegmatis ΔF6 vs. Wild type |
|
| Relations |
| BioSample |
SAMN14677111 |
| SRA |
SRX8161304 |
