title: NextSeq 500 sequencing; TCF/LEF dependent and independent regulation of Wnt/beta-catenin transcription
design: TCF/LEF dependent and independent regulation of Wnt/beta-catenin transcription
library_construction_protocol: Human embryonic kidney (HEK) 293T cells were cultured in media consisting of DMEM, high glucose (41966-029 Gibco) supplemented with 10% fetal bovine serum (Gibco), 1x penicillin‐streptomycin according to the manufacturer's recommendation. Two single-guide RNAs, targeting simultaneously either two exons or an exon and an intron of each TCF/LEF (see table) respectively were cloned separately into pSpCas9(BB)-2A-Puro (PX459) in order to sequentially ablate all full-length TCF/LEF isoforms. The constructs were co-transfected transiently in HEK293T cells using Fugene according to the manufacturer's recommendation and 24 hours after transfection puromycin selection was applied for an additional 48 hours. Cells were then split and seeded at clonal density. Clones from single cells were manually picked, and analyzed by Western blot for the expression of TCFs. The same approached was used also for the generation of the beta-catenin K.O. Table shows the gRNA sequence and the corresponding target region. RNA from HEK293T cells was extracted using TRIzol reagent (Ambion). Libraries were prepared with TruSeq RNA stranded library preparation.
attributes.tag: Experimental Factor: genotype
attributes.value: Tcf/Lef knockout of all four isoforms
attributes.tag: Experimental Factor: compound
attributes.value: CHIR 99021
attributes.tag: Experimental Factor: dose
attributes.value: 10