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Sample GSM7085183

Query DataSets for GSM7085183

Status

Public on Jul 06, 2023

Title

Vero76 cells, R. rickettsii st. Iowa, 4hpi, Rep3

Sample type

SRA

Source name

Vero76 (ATCC CCL-81)

Organism

Chlorocebus aethiops

Characteristics

cell line: Vero76 (ATCC CCL-81)
agent: R. rickettsii st. Iowa
time: 4 hour post infection

Treatment protocol

Vero76 cells in 6-well plates (2/replicate) were infected with R. rickettsii Morgan or Iowa at a multiplicity of infection (MOI) of 30 for a 4 hr time point and MOI of 1 for 24 and 48 hr time points.
Cultures were centrifuged at 2000 RPM for 5 min at room temperature to synchronize infections, and were incubated at 34ºC for 4, 24, or 48 hr prior to harvesting by scraping into Trizol. Purified rickettsiae of each strain were also extracted into Trizol. All conditions were performed in triplicate.

Growth protocol

Vero76 cells (ATCC® CCL-81™) were grown in RPMI medium plus 5 % heat-inactivated fetal bovine serum (FBS) at 37˚C in a 5% CO2 atmosphere.

Extracted molecule

total RNA

Extraction protocol

RNA containing aqueous phase was extracted using Qiagen AllPrep DNA/RNA 96-well system (Valencia, CA). The RNA yield was determined by spectrophotometry at 260 nm and 280nm and the integrity was assessed using the Agilent 2100 Bioanalyzer using RNA 6000 Pico kit (Agilent Technologies, Santa Clara, Ca). The average RNA Integrity Number (RIN) was 9.2.
Ribo-Zero Epidemiology kits (Illumina, Inc, San Deigo, CA) were used to deplete the samples of ribosomal RNAs prior to library preparation with the TruSeq Total RNA-Seq Library Prep Kit (Illumina), starting at the Elute-Frag-Prime step without further modification. Final libraries were assessed on a BioAnalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA) and quantified using the Kapa Quantification Kit for Illumina MiSeq (v3 chemistry )Sequencing (Roche, Basel, Switzerland).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

12703

Data processing

RNA-Seek (https://github.com/OpenOmics/RNA-seek)
A custom hybrid reference genome database was built with Chlorocebus sabaeus (Green Monkey) reference genome (GCA_000409795.2) – background of Vero cells, and R. rickettsii str. Iowa genome (GCA_000017445.3) using ‘build’ function in RNA-Seek pipeline.
Initial data preprocessing included read quality check with FASTQC v0.11.5, screening for sources of contamination (FQScreen v0.9.3, Kraken v2.0.8), removal of adapters and short sequences, and quality trimming with Cutadapt v1.18.
Preprocessed reads were then mapped to the hybrid genome reference using STAR v2.7.6a aligner, and reads mapping to C. sabaeus and R. rickettsia genes were quantified using RSEM v1.3.0.
The gene quantification data was split between C. sabaeus and R. rickettsia genes. The R. rickettsia gene expression data was further differential gene expression analyses.
Assembly: Hybrid genome (Chlorocebus sabaeus + R. rickettsii str. Iowa)
Supplementary files format and content: tab-delimited text files include RPKM counts (non-normalized) for R. rickettesia strain Iowa genes

Submission date

Mar 07, 2023

Last update date

Jul 06, 2023

Contact name

Neelam Redekar

Organization name

NIH

Department

NIAID

Street address

9000 rockville pike