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Sample GSM4634580

Query DataSets for GSM4634580

Status

Public on Dec 07, 2020

Title

NC19_HKSA

Sample type

SRA

Source name

PBMC

Organism

Homo sapiens

Characteristics

patient: NC19
carrier status: Non-carrier
age: 11 years old
Sex: Female
libraryid: lib2772
treatment: HKSA

Treatment protocol

PBMCs were stimulated for 6 h with (1) heat-killed S. aureus (10e7 cells/ml, InvivoGen), (2) a mixture (10e7 cells/ml) of all S. aureus isolates obtained from carrier subjects (“Carrier Mix”), or (3) un-stimulated, for the same time period. Due to the limited cell numbers that could be obtained from each pediatric donor, each treatment and control group/participant were carried out as singletons. After the incubation period, cells were washed with PBS and lysed using RLT buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma). Cell lysates were stored at -80°C until RNA extraction.

Growth protocol

Peripheral blood was drawn into ACD VacutainerÒ tubes from participants enrolled in the study using standard phlebotomy techniques. Specimen tubes were labeled and transported at 4°C to the University of Texas Health Science Center at Houston School of Public Health for PBMC isolation. PBMCs were isolated from whole blood within 4 h of collection using Ficoll Paque plus (GE Healthcare) and frozen down in 7% DMSO in fetal bovine serum and cooled at -1°C/min in a freezing container (Nalgene, ThermoFisher Scientific). Frozen PBMCs were stored at -80°C before shipping on dry ice to Benaroya Research Institute for further processing and stimulation.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from PBMCs using the RNeasy Mini Kit (Qiagen) and RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).
Sequencing libraries were constructed from total RNA using TruSeq RNA Sample Preparation Kits v2 (Illumina, San Diego, CA). Libraries were clustered onto a flowcell using a cBOT amplification system with a HiSeq SR v3 Cluster Kit (Illumina). Single-read sequencing was carried out on an Illumina HiScanSQ sequencer using a HiSeq SBS v3 Kit to generate 100-base single-end reads with a target of approximately 10 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiScanSQ

Data processing

The raw reads were processed in Galaxy using TopHat (with bowtie) for alignment (GRCh37 as reference genome), BAM-to-SAM, Picard Alignment Summary Metrics, and Picard RNAseq Metrics. Genes were quantified using htseq-count. Raw counts were used for downstream analysis and Fragments Per Kilobase Million (FPKM) values were used for plotting. FPKMs s for genes were obtained using edgeR
To identify differentially-expressed genes, a general linear model using negative binomial distribution with no random effects was applied using edgeR. Genes having a false discovery rate (FDR) <0.05 were considered differentially expressed.
Genome_build: GRCh37
Supplementary_files_format_and_content: text file including a matrix table with rpkm for every gene and every sample
Supplementary_files_format_and_content: text file including a matrix table with raw gene counts for every gene and every sample

Submission date

Jun 24, 2020

Last update date

Jan 03, 2024

Contact name

Stephanie Osmond

E-mail(s)

[email protected]

Organization name

Benaroya Research Institute

Street address

1201 9th Ave