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Sample GSM4295051

Query DataSets for GSM4295051

Status

Public on Apr 28, 2020

Title

siPAPα/γ_Rep_1

Sample type

SRA

Source name

iSLK cells

Organism

Homo sapiens

Characteristics

cell line: iSLK
pabpn1 and papalpha/gamma-mediated rna decay (ppd): yes (pathway inactive)
ars2-dependent decay: no (pathway active)

Treatment protocol

iSLK cells were transfected with 20 or 40 nM siRNA (Silencer Select, ThermoFisher) using RNAiMAX transfection reagent (Invitrogen) per manufacturer’s instruction. Specifically, we used final concentrations of 20 nM siRNAs for ARS2. For PAPα/γ, we used 20 nM each of siRNAs that target PAPα or PAPγ for a total of 40 nM siRNA. Twenty-four hours after siRNA transfection, cells were split into new plates and allowed to grow for another 24 hours, after which doxycycline and NaB was added to induce lytic reactivation. Thus, total RNA was harvested 72 hours post siRNA transfection and 24 hours post lytic reactivation.

Growth protocol

iSLK cells were grown at 37°C with 5% CO2 in DMEM (Sigma) supplemented with 10% Tet-Free fetal bovine serum (FBS, Atlanta Biologicals), 1x penicillin-streptomycin (Sigma), and 2 mM L-glutamine (Fisher). iSLK WT cells were grown in the presence of 0.1 mg/mL G418 (Fisher), 1 µg/mL puromycin (Sigma) and 50 µg/mL hygromycin.

Extracted molecule

total RNA

Extraction protocol

RNA was harvested using TRI reagent (Molecular Research Center, Inc.) according to the manufacturer’s protocol. Total RNA was purified using Chloroform-isopropanol extraction. RNA samples were quantified using Nanodrop (Thermofisher) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA).
RNA sequencing libraries were prepared using the KAPA Biosystems Stranded mRNA-Seq kit as per manufacturer’s protocol. Briefly, mRNAs were first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 6 minutes at 94 °C in the presence of magnesium. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition (NEBNext multiplex Oligos for Illumina) and library enrichment with limited cycle PCR. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

3_14
Poly-A RNA
processed data file: countTable.fpkm.csv

Data processing

The qualities of sequencing reads were evaluated using NGS QC Toolkit (v2.3.3) (Patel and Jain, 2012) and high-quality reads were extracted.
The human reference genome sequence and gene annotation data, hg19, were downloaded from Illumina iGenomes (https://support.illumina.com/sequencing/sequencing_software/igenome.html). The viral genome was downloaded from NCBI GenBank (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/nuccore/GQ994935.1). The qualities of RNA-sequencing libraries were estimated by mapping the reads onto human transcript and ribosomal RNA sequences (Ensembl release 89) using Bowtie (v2.2.9) (Langmead and Salzberg, 2012).
STAR (v2.5.2b) (Dobin et al., 2013) was employed to align the reads onto the human and viral genomes, Picard (v1.140) (https://broadinstitute.github.io/picard/) was employed to sort the alignments, and HTSeq Python package (Anders et al., 2015) was employed to count reverse-stranded reads per gene. DESeq2 R Bioconductor package (Gentleman et al., 2004; Anders et al., 2010) was used to normalize read counts and identify differentially expressed (DE) genes.
Genome_build: hg19 (GRCh37) and GQ994935.1
Supplementary_files_format_and_content: countTable.fpkm.csv: Comma-separated text file of viral gene fpkm counts.

Submission date

Feb 04, 2020

Last update date

Apr 28, 2020

Contact name

Nicholas K Conrad

E-mail(s)

nicholas.conrad@utsouthwestern.edu

Phone

2146480795

Organization name

UT Southwestern Medical Center