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Sample GSM2653746

Query DataSets for GSM2653746

Status

Public on Dec 04, 2017

Title

Fibro_∆UL138 24hpi

Sample type

SRA

Source name

Fetal Lung Fibroblast, ∆UL138 HCMV infected 24 hours

Organism

Human betaherpesvirus 5

Characteristics

host cell type: Fetal Lung Fibroblast MRC-5 (ATCC)
passage: 22
selection: None

Growth protocol

MRC-5 lung fibroblasts were maintained with DMEM (Gibco) supplemented with 10% FBS, 100 U/mL penicillin and streptomycin, 2mM L-alanyl-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 mM HEPES

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using a ZR-Duet DNA/RNA miniprep kit (Zymo Research, Irvine, CA)
RNA libraries were prepared for Illumina sequencing using either Agilent's SureSelect Strand Specific RNA Library Preparation Kit or Kapa Biosystem's KAPA Stranded mRNA-seq kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

∆UL138 HCMV infected 24 hours
Raw_Fb16.txt

Data processing

Raw sequencing data was de-multiplexed and fastq files generated using either built-in software (MiSeq) or CASAVA (HiSeq).
Raw sequence data were first evaluated using FastQC (v0.11.3, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and preprocessed for quality through a combination of trimming and filtering using Trim Galore (v0.4.0, 15 bases were trimmed off from the 5'-end of the reads and 5 bases were trimmed off from the 3'-end, Phred score threshold of 20 and minimum length of 50 bp, Paired, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Quality reads were then uniquely mapped to HHCMV (strain TB40/E, GenBank: EF999921.1) and human (GRCh38) genomes, respectively, both using Tophat2 (v2.1.1) with strand-specific alignment of fr-firststrand. For HHCMV alignment, the maximum intron size was set to 5kb as described on Tophat2 manual, and the uniquely mapped HCMV, but not human, reads were used for all subsequent analyses.
Raw read counts for each sample were obtained by mapping reads at the gene-level using HTSeq-count tool from the Python package HTSeq, with a stranded-reverse setting. DESeq2 R-package (v1.8.2) was then used to perform differential expression (DE) and statistical analysis, with a biological sample from SS and NS libraries of same cell donor grouped.
Raw read counts for genes over a group of six biological samples of fibroblast or CD34+ HPC infection were normalized through a regularized logarithm transformation (rlog) implemented in DESeq2, and kernel density estimates (KDE) were then obtained using the density R function with default parameters. The six density curves were overlaid on one plot. The featured density variation, associated with fibroblast or CD34+ HPC infection, was further evaluated using an extended group comprised of total four time points of post infection in fibroblasts (12, 24, 48, 72hpi, total 12 samples) and all NS/SS replicates from different cell donors of CD34+ HPC infection (total 24 samples). Density variation across samples was accessed by different bandwidth settings (0.5, 0.75, 1 and 1.25), which determine the degree of smoothing in the estimate of the density function of statistical software package R (http://www.r-project.org/). In addition, density variation across samples was assessed by the comparison between real and random samples, where raw read count for each gene in each sample was the mean value of 100 permutations of involved real samples and rlog normalization was performed.
Variability with respect to transcriptomes from infection in fibroblasts or CD34+ HPCs was assessed by the squared coefficient of variation (CV2), i.e., the variance in expression of rlog normalized counts (σ2) divided by the squared mean expression (μ2) and then multiplied by 100. The cumulative CV2 was also obtained as a final squared coefficient of variation representing the variability in different cell types.
In addition to rlog normalized counts, we quantified expression level of genes by merging annotated isoforms' FPKM values from Cufflinks (v2.2.1).
Genome_build: EF999921.1 (HCMV), GRCh38 (Human)
Supplementary_files_format_and_content: [Raw_Fb16.txt] Raw HCMV reads counts for fibroblast infection
Supplementary_files_format_and_content: [Raw_NSSS12.txt] Raw HCMV reads counts for CD34+ HPC infection from donor 1
Supplementary_files_format_and_content: [Raw_donor_NSSS14.txt] Raw HCMV reads counts for CD34+ HPC infection from donor 2 and donor 3
Supplementary_files_format_and_content: [Raw_natural.txt] Raw HCMV reads counts for natural infection
Supplementary_files_format_and_content: [FPKM13.txt] Abundance measurements of HCMV genes for CD34+ HPC infection (donor 1) and natural infection

Submission date

Jun 08, 2017

Last update date

May 15, 2019

Contact name

Janko Nikolich-Zugich

E-mail(s)

nikolich@email.arizona.edu

Phone

520-626-6065

Organization name

University of Arizona