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Sample GSM4567118

Query DataSets for GSM4567118

Status

Public on Mar 23, 2021

Title

S. aureus inoculum, Replicate 3

Sample type

SRA

Source name

ATCC 29213

Organism

Staphylococcus aureus

Characteristics

group: Inoculum
s. aureus strain: ATCC 29213

Treatment protocol

Microtiter plates were inoculated with 20 μl of bacterial stock suspension corresponding to a multiplicity of infection (MOI) of 16, followed by centrifugation at 300g for 5 min to sediment the bacteria. After 1½ hours post-infection (hpi), wells were supplemented with lysostaphin (10 μg/ml) + gentamicin (100 μg/ml). HUVEC samples were collected by washing 3 times in PBS followed by trypsination for 10 min, centrifugation, and washing the pellet followed by centrifugation and resuspension of the pellet in 100 μl PBS. Samples were then snap-frozen in liquid nitrogen and kept at -80°C until RNA isolation.

Growth protocol

The Staphylococcus aureus ATCC29213 reference strain and primary human umbilical cord endothelial cells (HUVEC from pooled donors, passage 2-3, PromoCell) were used for all experiments. Prior to experiments, the bacteria were grown until exponential growth phase in tryptic soy broth (2½h at 37°C), and bacterial stock solution could then be prepared by centrifugation and resuspension of pellet in phosphate buffered saline (PBS) followed by adjustment to OD600=0.1. HUVECs were grown until confluence in basal medium (Endothelial Cell Growth Medium 2, PromoCell) with fetal bovine serum (FBS) and penicillin-streptomycin (PS) and then split into microtiter plates. Prior to experiments, the confluent HUVEC microtiter plates were washed 3 times in PBS and filled with 0.5 ml basal medium+FBS without PS.

Extracted molecule

total RNA

Extraction protocol

Pellets were resuspended in 700 μl acetate buffer (20 mM NaOAc, 1 mM EDTA, 0.5% SDS, pH 4.5) and transferred to FastPrep tubes containing lysing matrix B. Cells were lysed in a Thermo Savant FastPrep FP120 Cell Homogenizer at 6.0 m/sec for 3 x 40 sec with 1 min pause on ice in between runs, and then centrifuged for 5 min at 20,000 g. Lysate was transferred to tubes containing 500 μl phenol solution (2:1 phenol in water adjusted to pH 4.5 with NaOAc) and 100 μl chloroform followed by incubation at 65 °C at 1100 rpm in a thermomixer for 15 min with vortexing every 2 min. After centrifugation, the aqueous phase was transferred into tubes containing 500 μl chloroform followed by vortexing, centrifugation, and transferring to new tubes. The RNA was precipitated using 96% EtOH and 10vol% 3M NaOAc on ice for ≥1 hour. Tubes were centrifuged for 60 min at 4°C, and pellets were washed in ice cold 70% EtOH followed by centrifugation for 10 min. Supernatant was removed and pellets were then air-dried and dissolved in sterile ddH2O. RNA quantity was measured using Nanodrop spectrophotometer, and RNA quality was then verified by fragment analysis using a Fragment Analyzer (Agilent), followed by RiboZero Gold (MRZE724, Epidemiology/Illumina) treatment on the 3 samples from each group to remove both human and bacterial rRNA.
After DNase treatment, library preparation was performed using the NebNext® UltraTM Library prep kit (NEB) followed by quality assessment by the fragment analyzer

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

4986
29213.tsv.gz

Data processing

Fastq files were preprocessed using cutadapt and base qualities checked using fastqc.
The reads were mapped against the human reference genome hg38 using STAR. Unmapped reads were mapped to the NC_007795.1 reference genome using bwa mem.
Gene read coverages were computed using featureCounts.
Differential gene expression analysis was conducted using the DESeq2 workflow with the BH corrected Wald test and log2FC skrinkage
Genome_build: hg38, NC_007795.1
Supplementary_files_format_and_content: Tab separated text files with gene read coverages.

Submission date

May 25, 2020

Last update date

Mar 23, 2021

Contact name

Christian Garde

E-mail(s)

[email protected]

Organization name

Evaxion Biotech

Street address

Dr Neergaards Vej 5F