mESC_KO_r3
Mus musculus
Protocols: mESCs were cultured on 0.2% gelatin coated plates in ES-DMEM medium as described previously. (Leeb, M., Perry, A. C. & Wutz, A. Establishment and Use of Mouse Haploid ES Cells. Current protocols in mouse biology 5, 155-185, doi:10.1002/9780470942390.mo140214 (2015).) To generate CRISPR/Cas9 Phf3 KO in mESCs, Rex1GFPd2::Cas9 (RC9) ES cells were used that carry a destabilized GFP-reporter for Rex1-expression and a stably expressed Cas9 transgene integrated into the Rosa26 locus. ES cells were co-transfected with 0.5 µg gRNA-expressing plasmid and 0.1 µg of dsRed expressing plasmid. After 2 days dsRed-positive cells were FACS-sorted and plated at clonal density into 60 mm TC-dishes. After 7 days colonies were picked and expanded in 96-well plates. To identify KO clones, genomic DNA was isolated and PCR-amplified Cas9 target region was sequenced. Total isolated RNA was first treated with recombinant DNaseI (Roche), cleaned up using peqGOLD PhaseTrap A tubes (Peqlab), and rRNA-depleted using the Ribo-Zero kit (Illumina). RNA-seq libraries samples were prepared using the NEBNext Ultra II directional RNA library preparation kit (NEB) according to the manufacturer's instructions.
ENA-FIRST-PUBLIC
2019-12-06T04:03:07Z
ENA-LAST-UPDATE
2018-12-13T17:19:28Z
External Id
SAMEA5175459
INSDC center name
Max Delbrueck Center
INSDC first public
2019-12-06T04:03:07Z
INSDC last update
2018-12-13T17:19:28Z
INSDC status
public
Submitter Id
E-MTAB-7526:mESC_KO_r3
broker name
ArrayExpress
cell line
E14
cell type
embryonic stem cell
common name
house mouse
developmental stage
embryo
disease
normal
genotype
Rex1GFPd2::Cas9; CRISPR/Cas9 mediated knockout of PH3
organism part
inner cell mass
phenotype
PH3 homozygous knockout
sample name
E-MTAB-7526:mESC_KO_r3
scientific_name
Mus musculus