Ad2_5_S3
Homo sapiens
Protocols: HEK293 cells were transfected using FuGENE HD with PiggyBac plasmid encoding trimethoprim stabilizable catalytically inactivated DDdCas9GFP under a doxycycline inducible promoter, PiggyBac rtTA-IRES-Neo, PiggyBac plasmid encoding EEA-motif targeting guide 1 (CCCAGCACTTTGGG) with Puro selection and PiggyBac transposase plasmid. CRISPR plasmids will be available on Addgene (https://www.addgene.org/Timo_Otonkoski/). Transfected cells were selected with puromycin and G418 and subcloned. Clones were selected for further experiments based on absence of GFP in non-induced state and homogenous GFP expression upon doxycycline and trimethoprim addition. Inducible HEK293 cell subclones containing DDdCas9GFP and EEA-g1 were treated for three days with doxycycline (1µg/ml) and trimethoprim (1mM) to express the dCas9 construct. Treated cells and non-treated control cells were dissociated as single cell suspension from four clones (C3, C5, D2 and E2). 50 000 cells from each sample were collected for further processing. Cells were cultured in DMEM + 10% FBS. Nuclei were isolated from 50,000 cells using mild lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl2, 3 mM MgCl2 and 0.1% IGEPAL) Library amplification required 12 cycles and was performed using Phusion HotStart Mastermix. Purified by AMPureXP beads.
ENA first public
2018-06-07
ENA last update
2017-12-14
External Id
SAMEA104430668
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
2017-12-14T17:33:07Z
INSDC status
public
Submitter Id
E-MTAB-6195:Ad2_5_S3
broker name
ArrayExpress
cell line
HEK293
cell type
epithelial cell
common name
human
developmental stage
late embryo
disease
normal
genotype
PB-tight-DDdCas9GFP-IRES-Neo; PB-CAG-rtTA-IRES-Neo; PB-EEA-g1-PGK-Puro
organism part
kidney
phenotype
wild type phenotype
sample name
E-MTAB-6195:Ad2_5_S3