GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7446516

Query DataSets for GSM7446516

Status

Public on Jun 07, 2023

Title

U251_NF2

Sample type

SRA

Source name

Brain

Organism

Homo sapiens

Characteristics

tissue: Brain
cell line: U251(MG)
cell type: Pleomorphic/astrocytoid
treatment: PBS

Treatment protocol

Cells were infected with ZIKV GZ01 with a multiplicity of infection (MOI) of 1 plaque-forming unit per cell and incubated for 1h at 37 °C. Next, the inoculum was removed and the cells were washed twice with phosphate-buffered saline (PBS) to eliminate the unbound virus. Complete culture medium was added to each well, and cells were incubated at 37 °C and 5% CO2.

Growth protocol

HTR8 cells and U251 cells were seeded in 24-well plates at 5 × 10^4 cells per well one day prior to infection.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA).
RNA libraries were prepared for sequencing using standard llumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

FPKM.U251_NF2

Data processing

Cutadapt software (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9) was used to remove the reads that contained adaptor contamination,(command line: ~cutadapt -a ADAPT1 -A ADAPT2 -o out1.fastq -p out2.fastq in1.fastq in2.fastq -O 5 -m 100).
And After removed the low quality bases and undetermined bases ,we used HISAT2 software (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.0.4) to map reads to the genome (for example:Homo sapiens Ensembl v96),(command line: ~hisat2 -1 R1.fastq.gz -2 R1.fastq.gz -S sample_mapped.sam).
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-1.3.4d.Linux_x86_64) with default parameters (command line: ~stringtie -p 4 -G genome.gtf -o output.gtf -l sample input.bam).
Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8.Linux_x86_64).
After the final transcriptome was generated, StringTie and ballgown(http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × exon_length(kB)]),(command line: ~stringtie -e -B -p 4 -G merged.gtf -o samples.gtf samples.bam).
Assembly: Homo sapiens, Ensembl v96
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes FPKM values for each Sample

Submission date

Jun 05, 2023

Last update date

Jun 07, 2023

Contact name

Qiqi Chen

E-mail(s)

chenqq53@mail2.sysu.edu.cn

Phone

15591822232

Organization name

Sun Yat-sen University