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Sample GSM4091293

Query DataSets for GSM4091293

Status

Public on Dec 20, 2020

Title

FPV-3

Sample type

SRA

Source name

feline kidney cells

Organism

Felis catus

Characteristics

cell type: feline kidney cell line
infection: Feline Panleukopenia Virus infected
tine: 48 hours after treatment

Treatment protocol

For construction of the six RNA-seq libraries, F81 cells were cultured in 75 cm² cell flasks and divided into two groups (3 flasks per group). One group was infected with FPV-BJ04 at a dose of 1×106 TCID50, the control group left uninfected. At 48h post-infection, FPV-BJ04 infected (FPV) and uninfected cells (Ctrl) were treated using Trizol reagent (Invitrogen, CA, USA) for the following total RNA extraction.

Growth protocol

F81 cells were obtained from the Cell Resource Center of the Shanghai Institute for Biological Science, Chinese Academy of Science, and maintained in Dulbecco′s Modified Eagle Medium (DMEM) (Gibco, CA) containing 5% fetal bovine serum and 1% penicillin-streptomycin at 37 ℃ with 5% CO2. FPV strain (FPV-BJ04) is a strain of FPV originally isolated from feces of infected cats in a pet′s hospital, Beijing, China. Initially, the full genome of this strain has been sequenced accurately in our laboratory.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0.
Approximately 10 ug of total RNA was used to deplete ribosomal RNA according to the manuscript of the Ribo-Zero™ Magnetic Gold Kit (Illumina, San Diego, USA). After removing ribosomal RNAs, the left RNAs were fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the . TruSeq Stranded Total RNA HT Sample Prep Kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing ( 2 x 150 bp) on an Illumina Hiseq 4000 at the (LC Sceiences,USA) following the vendor’s recommended protocol.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 4000

Description

lncRNA sequencing

Data processing

Illumina Casava1.8 software used for basecalling.
A cDNA library constructed by technology from the total RNA was sequenced run with Illumina sequence platform. Prior to assembly, the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20) were removed.
We aligned reads of samples to the reference genome using HISAT package.
The mapped reads of each sample were assembled using StringTie. Then, all transcriptomes from Samples were merged to reconstruct a comprehensive transcriptome using perl scripts.
Transcripts that overlapped with known mRNAs and shorter than 200 bp were discarded. Then we utilized CPC,CNCI to predict transcripts with coding potential. All novel transcripts with CPC score ≤-1 and CNCI score ≤0 and class code (I, j, o, u, x) were conside red as lncRNAs. StringTie and edgeR was used to estimate the expression levels of all mRNA and lncRNA

Submission date

Sep 23, 2019

Last update date

Dec 20, 2020

Contact name

Lingling Zhang

E-mail(s)

zll080504@163.com

Phone

15169117826

Organization name

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences