title: HEK_E2F6ko_IgG
organism: Homo sapiens
description: 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).]
attributes.tag: ENA first public
attributes.value: 2018-01-01
attributes.tag: ENA last update
attributes.value: 2017-08-24
attributes.tag: External Id
attributes.value: SAMEA104225646
attributes.tag: INSDC center alias
attributes.value: Institute of Molecular Biology and Tumor Resarch (IMT), Philipps-University of Marburg, Marburg, Germany
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Biology and Tumor Resarch (IMT), Philipps-University of Marburg, Marburg, Germany
attributes.tag: INSDC first public
attributes.value: 2018-01-01T17:01:42Z
attributes.tag: INSDC last update
attributes.value: 2017-08-24T15:23:14Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6006:HEK_E2F6ko_IgG
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: E2F6 knockout
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-6006:HEK_E2F6ko_IgG