Illumina HiSeq 1500 sequencing; PRC1.6 RNAseq
PRC1.6 RNAseq
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. RNAeasy Mini system (Qiagen) according to manufacturer's instructions TruSeq Stranded mRNA Library Prep Kit
Experimental Factor: genotype
wild type genotype