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Sample GSM4445356 Query DataSets for GSM4445356
Status Public on Jul 01, 2021
Title hESC_naive_brg-2
Sample type SRA
Source name Embryonic stem cells
Organism Homo sapiens
Characteristics cell line: WIBR3
cell type: Embryonic stem cells
culture condition: Naive
genotype: BRG knockdown
Treatment protocol Human ESCs were infected with the lentivirus in the presence of 6 ug/ml polybrene (Millipore). Infected cells were plated on mitomycin C inactivated DR4 feeder cells and selected with 1 ug/ml of puromycin for 5 days since 48 h after transduction.
Growth protocol Conventional (primed) human ESCs were maintained on mitomycin C inactivated mouse embryonic fibroblast (MEF) feeder layers and passaged using Collagenase (1 mg/mL) or manual methods. Primed human ESCs were cultured in human ESC medium (hESM) [DMEM/F12 (Invitrogen) supplemented with 15% fetal bovine serum (Gibco HI FBS, 10082-147), 5% KnockOut Serum Replacement (Invitrogen), 2 mM L-glutamine (MPBio), 1% nonessential amino acids (Invitrogen), 1% penicillin-streptomycin (Lonza), 0.1 mM β-mercaptoethanol (Sigma) and 4 ng/ml FGF2 (R&D systems)]. Naive human ESCs were cultured on mitomycin C-inactivated MEF feeder cells, and were passaged by a brief PBS wash followed by single-cell dissociation using 3-5 minute treatment with Accutase (Gibco) and centrifugation in fibroblast medium [DMEM (Invitrogen) supplemented with 10% FBS (Gibco HI FBS, 10082-147), 2 mM L-glutamine (MPBio), 1% nonessential amino acids (Invitrogen), 1% penicillin-streptomycin (Lonza) and 0.1 mM β-mercaptoethanol (Sigma)]. For conversion of pre-existing primed human ESC lines, we seeded 2 x 105 trypsinized single cells on a MEF feeder layer in hESM supplemented with ROCK inhibitor Y-27632 (Stemgent, 10 μM). 2 days later medium was switched to 5i/L/A or 4i/L/A (no IM12)-containing naive human ESC medium. Following an initial wave of cell death, naive colonies appeared within 10 days and were expanded polyclonally using Accutase (Gibco) on a MEF feeder layer. Naive human pluripotent cells were derived and maintained in serum-free N2B27-based media supplemented with inhibitors and cytokines. 500 mL of medium was generated by including: 240 mL DMEM/F12 (Invitrogen; 11320), 240 mL Neurobasal (Invitrogen; 21103), 5 mL N2 supplement (Invitrogen; 17502048), 10 mL B27 supplement (Invitrogen; 17504044), 10 μg recombinant human LIF (made in-house), 2 mM L-glutamine (MPBio), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), 1% penicillin-streptomycin (Lonza), 50 µg/ml BSA (Gibco Fraction V, 15260), and the following small molecules and cytokines: PD0325901 (Stemgent, 1 μM), IM-12 (Enzo, 1 μM or 0 µM), SB590885 (R&D systems, 0.5 μM), WH4-023 (A Chemtek or Selleckchem, 1 μM), Y-27632 (Stemgent, 10 μM) and Activin A (Peprotech, 10 ng/mL). All naïve hESC experiments were conducted in 5% O2, 5% CO2.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the E.Z.N.A. total RNA kit I.
Library construction was performed using the SMARTer Directional cDNA Library Construction Kit (Clontech, 634933). Libraries were sequenced on an Illumina HiSeq3000 1 × 50 platform.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
Description Naive hESCs shBRG1#2
Data processing Reads were aligned to the genome using TopHat (v2.0.10) and Bowtie2 (v2.1.0) with the default parameter settings.
Transcript assembly and differential expression analysis were performed using Cufflinks (v2.1.1). Assembling of novel transcripts was not allowed (-G), other parameters of Cufflinks followed the default setting.
Genome_build: hg38
Supplementary_files_format_and_content: FPKMs
Submission date Mar 30, 2020
Last update date Jul 01, 2021
Contact name Jianlong Wang
Organization name Columbia University Irving Medical Center
Department Department of Medicine
Lab Jianlong Wang
Street address 650 W. 168th St.
City New York
State/province New York
ZIP/Postal code 10032
Country USA
Platform ID GPL21290
Series (2)
GSE147750 OCT4 cooperates with distinct chromatin remodelers in naive and primed pluripotent states in human [RNA-Seq]
GSE147751 OCT4 cooperates with distinct chromatin remodelers in naive and primed pluripotent states in human
BioSample SAMN14486407
SRA SRX8030312

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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