HEL141_1_p13
Homo sapiens
Protocols: Human pluripotent stem cells were grown in E8 and Matrigel conditions. Stem cells were collected at passage 10-17 (CRISPRa iPSCs), passage 38 (SeV iPSCs) and passage 50 (H9 ESCs, WA09). Human foreskin fibroblasts (ATCC, CRL-2429) were grown in DMEM and FBS and collected to Trizol Reagent at passage 9. Sev induced pluripotent stem cells (HEL46.11) were reprogrammed using the Cytotune iPS reprogramming kit, according to manufacturer's instructions. HEL46.11 were initially cultured on MEF feeders in hES-medium for 10 passages, after which the cells were adapted to grow in E8 medium on Matrigel coated dishes. CRISPR iPSCs (HEL136, HEL139, HEL140, HEL141) were reprogrammed with episomal reprogramming plasmids using dCas9VPH activator containing P53 shRNA and combinations of OCT4, SOX2, KLF4, MYC, LIN28A and EEA-motif targeting guide plasmids (https://www.addgene.org/Timo_Otonkoski/). Plasmids were delivered to the cells with Neon transfection system with 16050V, 10ms and 3x pulse settings. Reprogramming was performed in 1:1 hES-medium/fibroblast medium until colony formation and hES-medium after that, supplemented with 250µM sodium butyrate. Reprogrammed iPS cells were cultured in E8 medium on Matrigel coated dishes. All cell lines were confirmed reprogramming episome negative by PCR (plasmids) or RT-PCR (SeV). hES-medium (KnockOut DMEM (Gibco) supplemented with 20% KO serum replacement (Gibco), 1% GlutaMAX (Gibco), 0.1 mM beta-mercaptoethanol, 1% nonessential amino acids (Gibco), and 6 ng/ml basic fibroblast growth factor (FGF-2; Sigma)). Fibroblast medium (Dulbecco's modified Eagle's medium (DMEM; Life Technologies) containing 10% fetal bovine serum (FBS; Life Technologies), 2mM GlutaMAX (Life Technologies), and 100 µg/ml penicillin-streptomycin (Life Technologies)). 100 µl of chloroform per 500 ml of sample was added and mixed with Trizol Reagent. After centrifugation (12 000 g for 15 min) the transparent upper phase was collected and RNA was further purified using NucleoSpin RNA kit (Macherey-Nagel). RIN values were measured by Bioanalyzer (Agilent) and total-RNA concentrations scaled to equal (10 ng/μl) using Qubit assay (Thermo Fisther Scientific). Bulk-RNA transcriptome analysis was performed by the STRT RNA-seq method (PMID: 22481528) with minor modifications (PMID: 26874359). Briefly, ten nanogram of high-quality input RNA was converted to cDNA and amplified to form an Illumina-compatible 46-plex library. ERCC RNA Spike-In Mix 1 (cat. 4456740; ThermoScientific) was used in 1000X dilution. In total, 25 PCR cycles were used, but as six base-pair unique molecular identifiers (UMIs) were applied, only the absolute number of unique reads was calculated per analyzed sample.
ENA first public
2018-06-07
ENA last update
2017-12-14
External Id
SAMEA104429533
INSDC center alias
Department of Biosciences and Nutrition, Karolinska Institutet
INSDC center name
Department of Biosciences and Nutrition, Karolinska Institutet
INSDC first public
2018-06-07T17:02:48Z
INSDC last update
2017-12-14T13:55:25Z
INSDC status
public
Submitter Id
E-MTAB-6185:HEL141_1_p13
broker name
ArrayExpress
cell line
HEL141.1
cell type
induced pluripotent stem cell
common name
human
developmental stage
neonate
disease
normal
genotype
pCXLE-dCas9VP192-T2A-EGFP-shP53; GG-EBNA-EEA-5guides-PGKPuro, GG-EBNA-KM-PP; GG-EBNA-OS-PP
organism part
dermis
phenotype
expression of p53 shRNA and OCT4, SOX2, KLF4, MYC, LIN28A and EEA-motif targeting guide plasmids
progenitor cell type
CCD-1112Sk
protocol
non-integrative CRISPRa plasmids
sample name
E-MTAB-6185:HEL141_1_p13