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Sample GSM4611111

Query DataSets for GSM4611111

Status

Public on Apr 23, 2021

Title

H. influenzae untreated (control) - rep1

Sample type

SRA

Source name

in vitro bacterial culture

Organism

Haemophilus influenzae

Characteristics

treatment: Untreated
strain: Haemophilus influenzae 86-028NP

Treatment protocol

Bacterial cultures were treated with 11 infection-related stress conditions and a control (Ctrl) sample not treated with any stress: acidic stress (As), pH 3-5 for 10 minutes; bile stress (Bs), 0.5% bile salts for 10 minutes; low iron (Li), 250 μM 2,2-dipyridryl for 10 minutes; hypoxia (Hyp), low oxygen for 4 hours; nutritional downshift (Nd), incubation in 1X M9 salts for 30 minutes; nitrosative stress (Ns), 250 μM Spermine NONOate for 10 minutes; osmotic stress (Oss), 0.5% NaCl for 10 minutes; oxidative stress (Oxs), 0.5-10 mM H2O2 for 10 minutes; stationary phase (Sp); temperature (Tm), 41°C for 20 minutes; varied virulence inducing condition (Vic).

Growth protocol

All bacterial strains were grown in their optimal growth medium and temperatures as indicated in laboratories specialized in each species. Three bacterial cultures were grown overnight and then subcultured to a new culture vial with 1:50-1:100 dilution. The cultures were grown until the exponential phase with an OD600 of 0.1-0.6 and exposed to the 11 stresses separately. The stress conditions, reagents used to generate them, and the exposure times are given in Table S1. Un-exposed cultures at exponential growth were used as controls for differential expression analysis. For nutritional downshift, the three bacterial cultures were spun down at 9000 rpm for 2 minutes, the supernatant removed, the pellets resuspended in 1X M9 supplemented with 0.1 M MgCl2 and 0.1 M CaCl2, and incubated for 30 minutes. For hypoxia, 2 ml screw cap tubes were filled with bacterial culture at exponential growth and the caps were screwed on without leaving space for air. The exposure to the stress conditions was stopped by adding 5% (final concentration) phenol:ethanol solution.

Extracted molecule

total RNA

Extraction protocol

One milliliter of triplicate bacterial cultures exposed to the stresses and un-exposed control culture were immediately pelleted by centrifugation at 9000 rpm at room temperature for 2 minutes after adding phenol:ethanol. The supernatant were removed and the pellets resuspended in 0.5 ml Trizol solution. For Gram-negative bacteria, the cells were homogenized in Trizol solution by pipetting up and down 15 times. For Gram-positive bacteria, culture suspensions in Trizol were transferred to previously cooled bead beater tubes containing 0.1-mm glass beads and treated with Mini-Beadbeater (Biospec Products Inc, USA) twice at a fixed speed for 45 seconds, and then cooled on ice for 1 minute between the treatments. Culture homogenates were incubated at room temperature for 5 minutes and then supplemented with 0.2 ml chloroform, thoroughly mixed by shaking 10 times, and incubated for 3 minutes. After centrifugation at 12 000 g at 4°C for 15 minutes, the aqueous upper phase was carefully transferred to new RNase free tubes. An equal volume of 99% ethanol was added to the transferred aqueous phase and isolation continued using the Direct-Zol RNA Miniprep Plus (Zymo Research, USA) RNA purification kit protocol. Total RNAs were eluted in RNase free water in RNase free tubes. The total RNA concentrations were measured using the Qubit BR RNA Assay Kit (ThermoFisher Scientific, USA) and RNA integrity confirmed on a 0.8% agarose gel in TBE buffer.
All rRNA-depleted RNA-seq library preparations were performed according to Shishkin et al.,2015 with minor modifications. RNAtag-Seq allows multiple library preparations in one tube, with initial tagging of total RNA samples with modified (5’P and 3’ddC) DNA barcoded adaptors, each harboring a unique 8 nt sequence used to demultiplex individual libraries after sequencing. We combined the library preparation of three biological replicates from 11 stress-exposed samples and the un-exposed control sample, resulting in 36 library preparations in one tube for each bacterial strain. Therefore, 36 unique barcoded adaptors were used to tag the 36 replicates, with every three barcodes used for samples from the same stress conditions in all bacterial species (Table S2). A total of 100 ng total RNA was used for each biological replicate. The total RNA was fragmented in 2X FastAP Thermosensitive Alkaline Phosphatase buffer for 3 minutes at 94°C, DNase treated, and dephosphorylated with a combination of TURBO DNase and Thermosensitive Alkaline Phosphatase in 1X FastAP buffer for 30 minutes at 37°C. Fragmented, DNase-treated, and dephosphorylated total RNAs were cleaned with a 2X reaction volume of Agencourt RNAClean XP beads. Cleaned total RNAs were incubated with 100 pmol of a unique DNA barcode adaptor at 70°C for 3 minutes, and then ligated with T4 RNA ligase 1 for 90 minutes at 22°C. The ligation was stopped and the enzyme denatured by the addition of RLT buffer. The 36 denatured ligation mixes were then pooled and cleaned in a Zymo Clean & ConcentratorTM-5 column according to the manufacturer’s 200 nt cut-off protocol. The RNAs were eluted in 32 l of RNase free water. The ribosomal RNA was depleted using the Ribo-ZeroTM Magnetic Gold Kit (Bacteria) according to the manufacturer’s instructions. The first strand cDNA of each pool was generated using an AffinityScript Multiple Temperature cDNA synthesis kit with 50 pmol of AR2 primer at 55°C for 55 minutes. The RNA was degraded by adding a 10% reaction volume of 1 N NaOH at 70°C for 12 minutes and the reaction neutralized with an 18% reaction volume of 0.5 M acetic acid. After cleaning the reverse transcription primers with a 2X reaction volume of RNAClean XP beads, the 3Tr3 adapter was ligated with T4 RNA ligase 1 at 22°C with overnight incubation. The second ligation was cleaned first with a 2X, and secondly 1.5X, reaction volume of RNAClean XP beads. The cDNA was then used as the template for PCR reaction with FailSafeTM PCR enzyme mix using 12.5 pmol 2P_univP5 as forward primers and 12.5 pmol ScriptSeqTM Index (barcode) PCR primers as reverse primers. The PCR cycles were as follows: 95°C for 3 minutes, followed by 12 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 68°C for 3 minutes, and then finishing at 68°C for 7 minutes. The PCR product was cleaned first with a 1.5X, and secondly 0.8X, reaction volume of AMPure beads and eluted in RNAse free water. The library concentrations were measured using the QubitTM dsDNA HS Assay Kit and the library insert size determined by the Agilent DNA 1000 Kit in a 2100 Electrophoresis Bioanalyser Instrument (Agilent, USA).
RNAtag-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

rRNA depleted total RNA
HINF_processed.txt

Data processing

The RNA-seq libraries generated by RNAtag-Seq were sequenced by either single end or paired end Illumina sequencing. Each library harboring a pool of 36 RNA samples was demultiplexed according to the unique 8 nt on the ligated barcode adaptors to separate reads from each replicate by CLC Genomic Workbench version 20.
RNA-seq reads we aligned to the reference genome with CLC Genomic workbench version 20
For differential gene expression analysis, the trimmed mean of M values (TMM) (Robinson and Oshlack, 2010) normalization method was used to normalize the sequencing depth of the individual libraries. For each bacterial species, the comparisons were performed between multiple groups and the control sample so that the full dataset could be used for fitting the generalized linear model.
Genome_build: CP025774, CP008706, NC_017846, NC_001318, NC_006350, NC_006351, NC_008787, NC_008770, NC_08790, NC_017316, NC_011601, NC_017633, NC_017721, NC_017722, NC_017723, NC_017724, NC_008253, C_019551, NC_007146, NC_011333, NC_000921, NC_009648, NC_009649, NC_009650,NC_009651, NC_009652, NC_009653, NC_002942, NC_003210, NC_009525, NC_002946, NC_008767, NC_002516, NC_016810, NC_017718, NC_017719, NC_017720, , CM001474, NC_002952, NC_002953, NC_005951, CP020462, CP020463, NC_004368, NC_008533, NZ_CP008776, NC_012925, NC_002505, NC_002506, NC_010465
Supplementary_files_format_and_content: Matrix table with TPM values for every gene and every sample for each species

Submission date

Jun 11, 2020

Last update date

Jun 08, 2021

Contact name

Kemal Avican

E-mail(s)

kemal.avican@umu.se

Organization name

Umea University

Department

Department of Molecular Biology