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Sample GSM4173979

Query DataSets for GSM4173979

Status

Public on Nov 19, 2019

Title

S. sonnei infected larvae 2

Sample type

SRA

Source name

pool of 20 S. sonnei infected zebrafish larvae

Organisms

Shigella sonnei; Danio rerio

Characteristics

strain: AB (zebrafish), 53G transformed with pFPV25.1 (S. sonnei)
infection: Shigella sonnei
incubation temperature: 28.5C
collection point: 24 hours post injection

Growth protocol

Larvae were obtained by natural spawning and maintained at 28.5C until 3 days post fertilisation in 0.5xE2 medium (7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO4, 75 µM KH2PO4, 25 µM Na2HPO4, 0.5 mM CaCl2, 0.35 mM NaHCO3). For injections, larvae were then anaestesised in 0.5xE2 medium supplemented with 200 ug/ml tricaine, and injected with bacterial resuspension buffer alone (2% polyvinylpyrrolidone, 0.5% phenol red in phosphate buffer saline). Injected larvae were then returned in E2 medium without anesthetic and maintained at 28.5C for 24h before collection. Bacteria for injections were firsts grown at 37C O/N from glycerol stocks on tryptic soy agar plates containing 0.01% Congo red and 100 ug/ml Carbenicillin. One single smooth colony accumulating Congo red was selected and cultured O/N in tryptic soy broth containing 100 ug/ml Carbenicillin at 37C with agitation. O/N culture was then diluted 50x in tryptic soy broth containing 25 ug/ml Carbenicillin and grown at 37C with agitation until culture reached an OD of 0.55-0.65 at 600 nm. Bacteria were then spun down, washed in PBS and resuspended at 7000 CFU/nl in bcterial resuspension buffer for injections.

Extracted molecule

total RNA

Extraction protocol

Samples were snap frozen at -80°C, then 100 μl of RNA protect bacteria reagent (Qiagen) was added, followed by mechanical trituration with a pestle blender. Samples were supplemented with 100 μl of 30 mM Tris-HCl/1 mM EDTA solution at pH = 8, 33 μl of 50 mg/ml lysozyme (Thermo Scientific), 33 μl of proteinase K >600 U/ml (Thermo Scientific) and shaken for 20 min RT. Lysis was completed by adding 700 μl of RTL buffer (Qiagen), 3 μl of 1 M dithiothreitol (Sigma-Aldrich) and mechanical disruption. Undigested debris were spun down 3 min 10000 rpm and the supernatant was supplemented with 500 μl of 100% ethanol prior loading onto RNeasy mini columns (Qiagen). From this step onwards, the manufacturer’s guidelines were followed for RNA purification. RNA quality and integrity were assessed by using NanoDrop and Non-denaturing agarose gel electrophoresis. For further quality check, RNA sequencing, library construction and reads count, samples were outsourced to Vertis Biotechnologie AG (Freising, Germany). Bacterial and host mRNA were enriched prior library preparation by using Ribo-Zero Gold rRNA Removal Kit (Epidemiology, Illumina, San Diego, California).
The rRNA depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase . The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 – 550 bp using a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pools were sequenced on an Illumina NextSeq 500 system using 75 bp read length.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

ssonnei_processed_data.tsv, zebrafish_processed_data.tsv

Data processing

Base-calling was perform online during the sequencing procedure with the Real-Time Analysis (RTA) software version 2.4.11 and System Suite Version 2.1.2.1.
Conversion of the bcl2 file to de-multiplexed gzipped fastq files was performed using the bcl2fastq script v. 2.18.0.12 provided by Illumina.
Quality and adapter trimming of fastq files was performed with trimmomatic-0.36.jar SE using parameters: -phred33;ILLUMINACLIP:adapter:2:30:10;SLIDINGWINDOW:4:15;LEADING:3;TRAILING:3;MINLEN:30
Mapping of the reads to the genomes was performed with bowtie2 using parameter --local
Read counting was performed with featureCounts version 1.5.0-p1
Genome_build: GRCz11 (Danio rerio), ASM28371v1 (S. sonnei 53G)
Supplementary_files_format_and_content: tab-separated values (TSV) files include geneID, gene name, gene description, log2foldchange (Deseq2), pvalue (Deseq2), padj (Deseq2), chromosome name, position (start, end and strand), length, reads and RPKM per each sample

Submission date

Nov 18, 2019

Last update date

Nov 20, 2019

Contact name

Vincenzo Torraca

E-mail(s)

vincenzo.torraca@lshtm.ac.uk

Phone

+447519097861

Organization name

LSHTM