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Sample GSM2913283

Query DataSets for GSM2913283

Status

Public on Dec 26, 2018

Title

Empty vector_2

Sample type

SRA

Source name

human fetal stem cells

Organism

Homo sapiens

Characteristics

cell type: control fetal neural stem cells (fNSCs)
passages: 9-15
transfection: empty vector

Treatment protocol

The empty pCAGIG and full-length E protein expressing pCAGIG vector were transfected in fNSCs at confluency of 70-80% using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Cells were harvested after 24 hours of transfection.

Growth protocol

Telencephalon of 10-15 week-old human aborted fetus was used to isolate Neural stem cells (NSCs). Isolated fNSCs were cultured in poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) coated culture dishes in neurobasal media (Invitrogen, San Diego, CA, USA). The media was supplemented with Neural Survival Factor-1 (Lonza, Charles City, IA, USA), N2 supplement (Invitrogen), Bovine Serum Albumin (Sigma-Aldrich, St. Louis, MO, USA), glutamine (Sigma), 25 ng/ml basic fibroblast growth factor (bFGF) (Peprotech), 20 ng/ml epidermal growth factor (EGF) (Peprotech), penicillin and streptomycin solution (Invitrogen) and gentamicin (Sigma). Cells were passaged for at least 9 times and characterized before performing experiments. fNSCs were maintained in sterile humidified chamber at 37°C and 5% CO2 in above cocktail of media.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from fNSCs using the TRIzol reagent (Invitrogen/Life Technologies) following the manufacturer’s protocol. RNA concentration was quantitated using the Bioanalyzer.
SMARTer smRNA-Seq Kit for Illumina (Clonetech) was used for preparing libraries for the samples. The workflow involves polyadenylation, cDNA synthesis, and PCR amplification steps resulting in indexed cDNA library, followed by library purification, validation, gel-free bead based size selection and post-size selection library validation. The resulting validated cDNA indexed libraries have been sequenced on Illumina NextSeq 500 to generate ≥20 million reads per sample (paired-end analysis).

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NextSeq 500

Description

Small RNA sequencing.
Human fetus-derived stem cells.
Highly proliferating fNSCs.
Control fNSCs were transfected with empty pCAGIG for 24 hours (n=2).
Vec - VECTORP14_T (GE) - RPKM
processed data file: Vec_vs_S1P.txt

Data processing

Sequenced data has been processed to generate FASTQ files. Quality of fastq reads was checked using fastQC tool set in CLC bio Genomic Workbench ver 9.0 (CGWB). A minimum of 70% of the read pairs are of Q30 value.
Raw data for each sample were aligned to human microRNAnome database (miRBase release 21) with remaining reads aligned to the most recent human genome GRCh38 build in order to identify previously unknown regions that may encode for unique miRNAs. Pairwise comparison of the alignment results was done for identification of miRNAs that are differentially expressed at a significant level, i.e. up- or down-regulated.
Genome_build: GRCh38
Supplementary_files_format_and_content: Vec_vs_S1P.txt: Tab-delimited text file includes Reads Per Kilobase per Million (RPKM) for each sample.

Submission date

Jan 04, 2018

Last update date

Dec 26, 2018

Contact name

Reshma Bhagat

E-mail(s)

reshuubhagat@gmail.com

Phone

9717261061

Organization name

National Brain Research Centre