|
| Status |
Public on Jan 30, 2023 |
| Title |
bulk RNA SP1380 ssRA mutant replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial culture
|
| Organism |
Streptococcus pyogenes |
| Characteristics |
tissue: Bacterial culture
cell line: SP1380 ssRA
cell type: Bulk
growth phase: Late logarithmic
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Bacteria were statically grown to late-logarithmic growth phase in Todd Hewitt broth supplemented with 1% yeast extract (THY) at 37˚C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNeasy Plus Kit (Qiagen). Briefly, bacteria were grown to late-logarithmic growth phase in THY. Two volumes of RNAprotect (Qiagen) were added to the cultures and the samples were then centrifuged at 5,000 x g for 25 min at 4°C to pellet cells. RNA was isolated from the dry pellet as per the manufacturer’s instructions with an additional mechanical lysis step using Lysing Matrix B tubes (MP Biomedicals) on the FastPrep-2 5G bead beating grinder and lysis system (MP Biomedicals). To ensure complete removal of DNA, the RNA was then further purified using the TURBO DNA-free kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
RNAseq analysis was performed at the Australian Centre for Ecogenomics (University of Queensland, Brisbane, Australia). cDNA libraries were prepared from total RNA using TruSeq Stranded Total RNA Library Prep with Ribo-Zero Plus rRNA Depletion kit (Illumina). Sequencing of the cDNA libraries was performed on the NovaSeq 6000 System (Illumina) on a 2x150 bp SP flowcell run generating an average of 20 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Bulk RNA-seq from SP1380 ssRA mutant strain
|
| Data processing |
Size filtering using cutadapt verison 2.8, special parameters: -n 0 -U 0 --action none -m 45
rRNA read filtering using database comprised of rRNA from S. pyogenes SF370, 5448, and HKU488 and SortMeRNA version 4.3.4, special parameters: --fastx
Alignment of reads against SP1380 (CP060269) using BWA-mem, bwa version 0.7.17
Read counting using FeatureCounts, subreads version 2.0.0, special parameters: -s 1 --largestOverlap -B -t gene -g ID
Differential expression analysis using Deseq2 v.1.32.0 in R v. 4.0.0. SP1380 mutants compared invididually to SP1380 WT
Assembly: CP060269
Supplementary files format and content: SP1380_count_file.tsv - Tab seperated file - Count of SP1380 wildtype and mutants against SP1380 reference genome
Supplementary files format and content: SP1380_WT_v_ssrA.tsv - Tab seperated file - Differential gene expression results comparing SP1380 wild-type to ssRA mutant
Supplementary files format and content: SP1380_WT_v_rofA.tsv - Tab seperated file - Differential gene expression results comparing SP1380 wild-type to rofA mutant
|
| |
|
| Submission date |
Aug 29, 2022 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Magnus Ganer Jespersen |
| Organization name |
The University of Melbourne
|
| Street address |
792 Elizabeth street
|
| City |
Parkville |
| State/province |
Victoria |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL28678 |
| Series (2) |
| GSE212241 |
Characterisation of the genetic mutation driving enhanced superantigen SpeA expression in Streptococcus pyogenes M1UK (second SP1380) |
| GSE212243 |
Characterisation of the genetic mutation driving enhanced superantigen SpeA expression in Streptococcus pyogenes M1UK |
|
| Relations |
| BioSample |
SAMN30559718 |
| SRA |
SRX17302347 |
