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Sample GSM4721334

Query DataSets for GSM4721334

Status

Public on Aug 11, 2020

Title

TJ-1# nESCs rep1

Sample type

SRA

Source name

pluripotent stem cells

Organism

Homo sapiens

Characteristics

cell type: Embryo-derived pluripotent stem cells
passages: 10-15
genotype/variation: Wild type

Growth protocol

For cell culture, hiF-T were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 2mM GlutaMAX (Millipore). Primed ESCs/iPSCs (pESCs/piPSCs) were cultured in hESM containing DMEM/F12 (Gibco) with 20% KnockOut SR (Gibco), 1% nonessential amino acids (Millipore), 2mM GlutaMAX (Millipore), Penicillin Streptomycin(Millipore),8 ng/ml bFGF (Peprotech). Naïve ESCs/iPSCs (nESCs/niPSCs) were cultured in 5iLAF medium containing DMEM/F12: Neurobasal (1:1) (Gibco), 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 0.5% KnockOut SR (Gibco), 1% nonessential amino acids (Millipore), 2mM GlutaMAX (Millipore), Penicillin Streptomycin(Millipore), 20ng/ml human LIF (Milipore), 8 ng/ml bFGF (Peprotech), 50 μg/ml BSA (Sigma) and the following cytokines and small molecules: PD0325901 (Selleck, 1 μM), SB590885 (Selleck, 0.5 μM), WH-4-023 (Selleck, 1 μM), Y-27632 (Selleck, 10 μM), and Activin A (Peprotech, 20 ng/ml) and passaged by Accutase (Sigma) every 4-5 days as previously reported. All hESCs were routinely cultured at 37 °C in 5% CO2, 5% O2.

Extracted molecule

total RNA

Extraction protocol

For mRNA-seq, total RNAs were isolated from cells using TRizol (Invitrogen). To generate RNA sequencing libraries, KAPA Stranded mRNA-Seq Kit (KAPA) was used following the manufacturer’s instructions. For single cell RNAseq, cell clones were mechanically picked and washed with PBS supplemented with 0.5% BSA. Following the Covaris DNA shearing protocol for Smart-seq sequence library generation as previously described. Briefly, cells were lysed, RNAs with a polyadenylated tail were captured, reverse transcripted and preamplified.
RNA libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

PolyA RNA

Data processing

All sequenced reads were checked for quality control by using FastQC (v0.11.7) with default parameters.
Sequenced reads was removed adapters and low-quality sequence by using trim_galore (v0.4.2 ) with the default parameters.
The processed RNA-Seq reads were mapped to hg19 genome using STAR (v0.6.0). RefSeq genes were quantified to FPKM using Stringtie.
The processed RIP-Seq reads were mapped to hg19 genome using TopHat (v2.1.1) with default parameters.
FPKM for each sample and log2 fold change were calculated by using cuffdiff (v2.1.1).
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *fpkm: Tab-delimited text files include FPKM values.

Submission date

Aug 11, 2020

Last update date

Aug 12, 2020

Contact name

Bi Yan

E-mail(s)

1610807@tongji.edu.cn

Phone

18916058932

Organization name

Tongji university