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Sample GSM4279876

Query DataSets for GSM4279876

Status

Public on Feb 26, 2020

Title

Sample 10_nhp1_4_inf_raw

Sample type

SRA

Source name

Skeletal muscle infected tissue section

Organisms

Streptococcus pyogenes; Macaca fascicularis

Characteristics

strain: MGAS2221 (infected NHP skeletal muscle tissue)
growth condition: In vivo
replictaes: in vivo infected NHP 1-Section 4
growth condition/time of isolation: Collected 24 h post-inoculation

Treatment protocol

At the desired growth stage, mid-exponential (OD 600nm 0.5) or early-stationary (OD 600nm 1.55), the culture was treated with RNAprotect Bacteria Reagent and cells were harvested by centrifugation. For the in vivo growth the excised tissue sections were submerged in RNAShield and frozen at -80°C until processed.

Growth protocol

For in vitro growth, cells were grown in Todd-Hewitt broth supplemented with yeast extract (THY media) at 37° C in atmosphere supplemented with 5% CO2. For in vivo growth, cells were inoculated in the quadriceps muscle of three NHPs, and 24 h after infection the muscle was removed en bloc, and sections corresponding to the infection site were collected.

Extracted molecule

total RNA

Extraction protocol

In vitro grown pelleted cells were mechanically disrupted using silica beads, RNA was isolated using RNeasy kit, contaminating DNA was degraded using Turbo DNA-free (Ambion), and rRNA was depleted using Ribo-Zero (Illumina). The tissue (80-100 mg) corresponding to the in vivo samples was washed in PBS, homogenized, and mechanically disrupted using 0.1 mm and 0.5 mm beads. RNA was extracted (MasterPure Complete DNA and RNA purification kit, Lucigen), subjected to three consecutive DNase treatments with Turbo DNA-free (Ambion), and depletion of ribosomal RNA was performed with the Ribo-Zero Gold rRNA Removal Kit, Epidemiology (Illumina).
Resultant RNA from dahA mutant and parental wild-type samples was reverse transcribed to cDNA and sequencing libraries were generated using ScriptSeq RNA-seq preparation kit (Epicentre). cDNA libraries from in vivo samples were generated from rRNA depleted RNA using a NEBNext Ultra II Directional RNA Library Preparation kit for Illumina (NEB).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

1 x 108 CFU/kg serotype M1 strain MGAS2221 were inoculated in the skeletal muscle and tissue excised 24 h later
processed data: 'GAS_sections' spreadsheet in the 'processed_data.xlsx'

Data processing

Multiplexed single-end (SE) libraries were sequenced on an Illumina Illumina NextSeq500/550 instrument, basecalling and demultiplexing was done on the instrument, and the RNA-Seq data was output as fastq files.
RNA-seq reads from strains grown in vitro were mapped to reference strain MGAS2221 (GenBank accession number CP043530), and MGAS5005 (accession number NC_007297) using Edge-pro v1.3.1 (Estimated Degree of Gene Expression for Prokaryotes), and CLC Genomics workbench.
Sequencing read quality was evaluated with FASTQC software
Adapter contamination and read quality filtering was performed with Trimmomatic.
Reads were mapped to the genome of Macaca fascicularis (5.0, release-95, Ensembl (http://useast.ensembl.org/Macaca_fascicularis/Info/Annotation) using STAR. Reads mapping to the NHP genome were subsequently mapped to the GAS genome (MGAS2221, Genbank ID CP043530).
Cross-mapping reads were identified and excluded with SeqFilter (https://github.com/BioInf-Wuerzburg/SeqFilter/blob/master/README.org). Filtered reads were mapped to the genome of reference GAS strain MGAS2221 using EDGE-pro (http://ccb.jhu.edu/software/EDGE-pro/). Differential expression analysis was performed using DESeq2 version 1.16.1, or expression levels were normalized as RPKM (reads per kilobase of exon model per million reads) using CLC Genomics workbench.
Genome_build: Macaca fascicularis (5.0, release-95, Ensembl (http://useast.ensembl.org/Macaca_fascicularis/Info/Annotation)
Supplementary_files_format_and_content: Raw reads were normalized as a part of DESeq2 pipeline, as a part of CLC Genomics workbench, or using TPM are provided along with raw data.

Submission date

Jan 22, 2020

Last update date

Feb 26, 2020

Contact name

Priyanka Kachroo

E-mail(s)

pkachroo@houstonmethodist.org

Organization name

Houston Methodist Research Institute

Street address

6670 Bertner Avenue