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Sample GSM8042017

Query DataSets for GSM8042017

Status

Public on Jul 30, 2024

Title

RP19_cells, infected, 72hrs, rep1

Sample type

SRA

Source name

MDTC-RP19

Organism

Meleagris gallopavo

Characteristics

cell line: MDTC-RP19
cell type: B lymphocytes
genotype: WT
treatment: Infected
time point: 72hrs

Treatment protocol

A commercially available THEV live vaccine was purchased from Hygieia Biological Labs. The stock virus was titrated using an in-house qPCR assay with titer expressed as genome copy number(GCN)/mL, similar to Mahshoub et al (https://doi.org/10.1016/j.jviromet.2016.11.002) with modifications. Cells were infected in triplicates at a multiplicity of infection (MOI) of 100 GCN/cell, incubate at 41C for 1 hour, and washed three times to get rid of free virion particles. Samples in triplicates were harvested at 4-, 12-, 24-, and 72-h.p.i for total RNA extraction and RNA-sequencing

Growth protocol

Mock and infected MDTC-RP19 cells were grown as suspension cultures in 1:1 complete Leibovitz's L-15/McCoy's 5A medium with 10% fetal bovine serum (FBS), 20% chicken serum (ChS), 5% tryptose phosphate broth (TPB), and 1% antibiotics solution (100 U/mL Penicillin and 100ug/mL Streptomycin), at 41C in a humidified atmosphere with 5% CO2. When infected or mock infected, cells were maintained in 1:1 serum-reduced Leibovitz's L15/McCoy's 5A media (SRLM) with 2.5% FBS, 5% ChS, 1.2% TPB, and 1% antibiotics solution (100 U/mL Penicillin and 100ug/mL Streptomycin)

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from infected cells using Thermofishers' RNAqueous™-4PCR Total RNA Isolation Kit (#AM1914) per manufacturer's instructions.
Poly(A) RNA sequencing library was prepared following Illumina’s TruSeq-stranded-mRNA sample preparation protocol. 1ug of total RNA was used for this library construction

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Reads were trimmed with the Trim-galore (v0.6.6) program to achieve an overall Mean Sequence Quality of 36
Trimmed reads were mapped simultaneously to the complete genomic sequence of avirulent turkey hemorrhagic enteritis virus (THEV) and Meleagris gallopavo using Hisat2 (v 2.2.1), and reads mapping to THEV filtered as new BAM files with Samtools (v1.16.1)
StringTie (v 2.2.1) was used to assemble the transcriptome using a GTF file derived from a gff3 file obtained from NCBI, which contains the predicted ORFs of THEV as a guide. GFFCOMPARE (v0.12.6) was used to merge all transcripts from all time points without redundancy.
StringTie set to expression estimation mode was used to calculate FPKM scores for all transcripts after which Ballgown (ve2.33.0) in R was used to perform the statistical analysis on the transcript expression levels.
Regtools (1.0.0) was used to count all junctions, the reads supporting them, and extract all other information related to the junction.
Assembly: melGal5 and AY849321.1
Supplementary files format and content: tab-delimted text file containing the FPKM values for each sample
Supplementary files format and content: tab-delimted text file containing combined unique junctions from all replicates and time points

Submission date

Jan 28, 2024

Last update date

Jul 30, 2024

Contact name

Abraham Quaye

E-mail(s)

quayeabraham29@gmail.com

Phone

3852868146

Organization name

Brigham Young University