HEK_E2F6ko_antiE2F6
Homo sapiens
Protocols: HEK293 cells were cultured in DMEM/F-12 + GlutaMax medium (gibco) supplemneted with 10% fetal bovine serum (Sigma) and 1 % Penicillin/Strepomycin (Sigma) Generation of MGA-, E2F6-, L3MBTL2- and PCGF6-depleted cell lines HEK293 cells were transfected using FugeneHD (Promega, City, Country) with plasmids expressing mammalian-codon optimized Cas9 and sgRNAs targeting the coding region of human MGA, E2F6, L3MBTL2 or PCGF6. The parental vector pSpCas9-2A-Puro (pX459) was a gift from Feng Zhang (Addgene plasmid # 48139) . Sequence of oligonucleotides used for targeting: MGA gRNA6: CATCTGGAAAGGTACTCCCA gRNA7: GTCATACTTGAATTGTATAC E2F6 gRNA1: GGGTATTCTTGACTTAAACA gRNA2: GTTTAAGTCAAGAATACCCC gRNA3: GTCGATTCCATCTAAGACAT L3MBTL2 gRNA6: GGATGTGATGAAAGGGATGA gRNA7: GCCTCTGTCATCCAGACAGC PCGF6 gRNA3: GGTATGAAGACATTCTGTGA gRNA4: TGTACTACTATATTGCATTT The empty pX459 vector was transfected as a negative control. Puromycin selection (3 μg/ml) was carried out 48 hours after transfection for 3 to 6 days. Individual colonies were isolated and the targeted loci were genotyped by PCR and sequenced. Cell clones with indels in the targeted locus were further analyzed by Western blotting. ChIP was performed using the One Day ChIP Kit (Diagenode) according to manufacturer's instructions. Three to four individual ChIPs were pooled and purifed on QIAquick columns (Qiagen). Five nanograms of precipitated DNA were used for indexd deep sequency library preperation using the Microplex library preparation kit v2 (Diagenode). Libraries were purified on AMPure magnetic beads (Beckman Coulter).]
ENA first public
2018-01-01
ENA last update
2017-08-24
External Id
SAMEA104225643
INSDC center alias
Institute of Molecular Biology and Tumor Resarch (IMT), Philipps-University of Marburg, Marburg, Germany
INSDC center name
Institute of Molecular Biology and Tumor Resarch (IMT), Philipps-University of Marburg, Marburg, Germany
INSDC first public
2018-01-01T17:01:42Z
INSDC last update
2017-08-24T15:23:14Z
INSDC status
public
Submitter Id
E-MTAB-6006:HEK_E2F6ko_antiE2F6
broker name
ArrayExpress
cell line
HEK293
cell type
epithelial cell
common name
human
developmental stage
embryo
disease
normal
genotype
E2F6 knockout
organism part
kidney
sample name
E-MTAB-6006:HEK_E2F6ko_antiE2F6