CTRL1
Homo sapiens
Protocols: Passage 10 fibroblasts (ATCC, CRL-2429) were reprogrammed with CRISPR-mediated gene activation. Cells were electroporated with pCXLE-dCas9VP192-GFP-shP53 activator and combinations of guide plasmids, GG-EBNA-OSKML-PP, GG-EBNA-KM-PP and GG-EBNA-EEA-5guides-PP, using Neon transfection system with 1650V, 10ms and 3x pulse settings. 250k (day 4 samples) to 125k cells (day 8 and 12 samples) were plated on Matrigel coated 6-well culture plates per well. Cells were grown for four days in DMEM and 10% FBS after which culture medium was changed to 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)) supplemented with 250µM sodium butyrate. Cells were collected to Trizol Reagent on days 4, 8 and 12 after electroporation. Human foreskin fibroblast controls were collected from untreated cells cultured in DMEM and 10% FBS. In addition to the activator, samples marked 36bp contain guides targeting the 36bp EEA-motif, Samples marked OMKSL contain one plasmid with OCT4, SOX2, KLF4, MYC and LIN28A targeting guides and one plasmid with KLF4 and MYC targeting guides. Samples marked OMKSL+36bp contain OCT4, SOX2, KLF4, MYC and LIN28A targeting guide plasmid, KLF4 and MYC targeting guide plasmid and a 36bp EEA-motif targeting guide plasmid. These plasmids will be available on Addgene (https://www.addgene.org/Timo_Otonkoski/). Passage 10 fibroblasts (ATCC, CRL-2429) were reprogrammed with CRISPR-mediated gene activation. Cells were electroporated with pCXLE-dCas9VP192-GFP-shP53 activator and combinations of guide plasmids, GG-EBNA-OSKML-PP, GG-EBNA-KM-PP and GG-EBNA-EEA-5guides-PP, using Neon transfection system with 1650V, 10ms and 3x pulse settings. 250k (day 4 samples) to 125k cells (day 8 and 12 samples) were plated on Matrigel coated 6-well culture plates per well. Cells were grown for four days in DMEM and 10% FBS after which culture medium was changed to 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)) supplemented with 250µM sodium butyrate. Cells were collected to Trizol Reagent on days 4, 8 and 12 after electroporation. Human foreskin fibroblast controls were collected from untreated cells cultured in DMEM and 10% FBS. In addition to the activator, samples marked 36bp contain guides targeting the 36bp EEA-motif, Samples marked OMKSL contain one plasmid with OCT4, SOX2, KLF4, MYC and LIN28A targeting guides and one plasmid with KLF4 and MYC targeting guides. Samples marked OMKSL+36bp contain OCT4, SOX2, KLF4, MYC and LIN28A targeting guide plasmid, KLF4 and MYC targeting guide plasmid and a 36bp EEA-motif targeting guide plasmid. These plasmids will be available on Addgene (https://www.addgene.org/Timo_Otonkoski/). 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
2017-11-15
External Id
SAMEA104388808
INSDC center alias
Department of Biosciences and Nutrition, Karolinska Institutet
INSDC center name
Department of Biosciences and Nutrition, Karolinska Institutet
INSDC first public
2018-06-07T17:02:47Z
INSDC last update
2017-11-15T13:49:59Z
INSDC status
public
Submitter Id
E-MTAB-6186:CTRL1
broker name
ArrayExpress
cell line
CCD-1112Sk
cell type
fibroblast of dermis
common name
human
disease
normal
genotype
wild type genotype
organism part
dermis
phenotype
wild type phenotype
sample name
E-MTAB-6186:CTRL1
time
0