|
| Status |
Public on Aug 05, 2015 |
| Title |
M23NDcovS-LP-2 |
| Sample type |
SRA |
| |
|
| Source name |
cell pellets
|
| Organism |
Streptococcus pyogenes |
| Characteristics |
strain: M23ND
genotype: M23ND/covS-
growth: Logarithm phase
biological replicate: Replicate 2
|
| Treatment protocol |
The cells were digested with mutanolysin (500u/ml) in 300lm spheroplasting buffer (20mM Tris-HCl pH6.8, 10mM MgCl2, 56% raffinose) and incubated with 100mg/ml chloramphenicol at 37’C for 60 minutes.
|
| Growth protocol |
covS- mutant strain and the isogenic covS+ derivative were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY; BD-Difco) at 37°C under aerobic conditions. Cells were collected at logarithm phase (OD600=0.6) and stationary phase (OD600=1.0).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellets were harvested by centrifuge at 10,000 rpm for 5 minutes. Total RNA was isolated and purified from the cell pellets using the DNeasy Blood & Tissue Kit (GIAGEN) according to the manufacture’s protocol.
The library was constructed using the Epicentre ScriptSeq v2 Complete Kit (Bacteria) and protocol companion (Illumina, Inc., San Diego, CA). Briefly, the total RNA was treated with rRNA-depletion using the Ribo-Zero rRNA removal solution specifically for gram-positive organisms. The remaining RNA was purified using Agencourt AMPure XP Beads, then heat fragmented at 85°C for 5 minutes. The ends of the fragments were reparied and ligated with adapters. The ligated fragments were PCR amplified and library fragments of ~200 bp band were isolated from the gel. The pair-end 2x79bp sequencing was performed using the Illlumina Miseq platform and Miseq Reagent Kit v3 (Illumina, Inc).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8
The reads were aligned to the annotated genome of S. pyogenes M23ND using BWA v0.7. Only uniquely mappable reads were retained.
The expression levels were evaluated by counting the reads uniquely mapping to the encoding regions of the annotated genes in the genome and calculated as RPKM by normalizing to the total uniquely mappable reads and gene lengths.
The gene differential expression was evaluated using DESeq package in R environments. The negative binomial test was used to assess the significance p-value of the differential expression and Benjamini-Hochberg procedure was used for multiple test correction.
Genome_build: CP008695 (Streptococcus pyogenes M23ND)
Supplementary_files_format_and_content: Excel file for the gene expression values and differential expression.
|
| |
|
| Submission date |
Apr 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Yunjuan Bao |
| Organization name |
University of Notre Dame
|
| Street address |
1234 Notre Dame Avenue
|
| City |
Notre Dame |
| State/province |
Indiana |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL19982 |
| Series (1) |
| GSE67533 |
Transcriptome study of the covS-regulation of a Group A Streptococcus pyogenes M23ND |
|
| Relations |
| BioSample |
SAMN03458337 |
| SRA |
SRX977331 |
