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Sample GSM7771944

Query DataSets for GSM7771944

Status

Public on Aug 02, 2024

Title

WT mouse infected-2

Sample type

SRA

Source name

Skin

Organism

Staphylococcus aureus

Characteristics

disease state: control
tissue: Skin
genotype: C57Bl/6
host: mouse

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from punch biopsy homogenates of 1-day-old infection sites using the Direct-zol RNA extraction kit (Zymo), after treatment for 5 min with 50 μg/ml of lysostaphin. Isolated RNA was further treated with DNase according to the manufacturer’s instructions (Ambion).
A quantitative PCR (qPCR) assay was performed on the captured cDNA samples to determine the correct number of cycles required to generate the final sequencing library as well as to assess the efficacy of normalization and enrichment (73). Typically, higher threshold cycle (CT) values indicate greater depletion of host transcripts. The final library was created by PCR amplifying the samples with the full-length sequencing adaptors and 6-mer ScriptSeq (Epicentre) barcodes. Following PCR, the samples were cleaned and size selected in a two-step cleanup using 0.75 volumes of AMPure XP beads followed by 0.15 volumes to achieve a final library with an average size of ∼330 bp. Libraries were combined with 12 samples each, in equal amounts, and concentrated using DNA Clean & Concentrator-5 (Zymo Research). Prior to sequencing, the final libraries were quantified using qPCR.
Combined libraries were sequenced on an Illumina NextSeq instrument using 75-cycle kits. Samples were loaded at 1.8 pM, and 75-base single-end reads were obtained.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

wt2

Data processing

For bacterial sequence information, read abundance was quantified using the S. aureus USA300 FPR3757 reference genome (GenBank accession number NC_007793) and Kallist, a kmer-based pseudoalignment tool, with analysis and visualization using Degust (https://github.com/drpowell/degust). Degust uses Voom/Limma and generates an interactive website to analyze and explore the data. Differential expression was considered statistically significant by applying threshold cutoffs of a >1.5-fold change and a P value of <0.05 (adjusted for false discovery). Bacterial genes were categorized into orthologous groups, and pathways were interpreted using Genome2D.
Assembly: S. aureus USA300 FPR3757 reference genome (GenBank accession number NC_007793)
Supplementary files format and content: all genes.xls (differential expression results)

Submission date

Sep 11, 2023

Last update date

Aug 02, 2024

Contact name

Kunal Poorey

E-mail(s)

kpoorey@sandia.gov

Phone

9256675923

Organization name

Sandia National Labs