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The relevant analysis information for this experiment and the associated data is contained in the accompanying RNotebooks which - if run alongside the bespoke .RData files - will reproduce the results in the associated publication. **Experimental details:** Wild-type or homozygous PIK3CA-H1047R iPSCs were seeded in 12-well plates all coated with Geltrex from the same lot (#2052962; diluted in DMEM/F12 lot #RNBH0692). Cells were processed for seeding at a ratio of 1:15 according to the standard maintenance protocol. One day after seeding, individual treatments were applied to triplicate wells. Briefly, cells were first washed twice with 2 and 1 ml of Dulbecco’s PBS (DPBS) to remove residual growth factors. The base medium for individual treatments was Essential 6 supplemented with 10 ng/ml heat-stable FGF2. This was combined with one of the following reagents or their diluent equivalents: 100 ng/ml NODAL (diluent: 4 mM HCl), 250 nM BYL719 (diluent: DMSO), 5 uM SB431542 (diluent: DMSO). Cells were snap-frozen on dry ice after 24, 48 and 72 h following a single DPBS wash. Individual treatments were replenished daily at the same time of day to limit temporal confounders. Cellular RNA was extracted using Zymogen's Direct-zol RNA Miniprep Kit, and 200 ng used for complementary DNA (cDNA) synthesis with Thermo Fisher’s High-Capacity cDNA Reverse Transcription Kit. Subsequent SYBR Green-based qPCRs were performed on 2.5 ng total cDNA. A 5-fold cDNA dilution series was prepared and used as standard curve for relative quantitation of gene expression. TBP was used as normalizer following confirmation that its gene expression remained unaffected by the tested conditions. Melt curve analyses were used to confirm amplification of a single product by each primer. All primers had amplification efficiencies 95%-105%. Individual samples were loaded in duplicate in 384-well plates. TaqMan hPSC Scorecards (384-well) were used according to the manufacturer’s instructions with the following modifications. From each sample diluted to 20 ng/ul, two 50 μl RT reactions were set up, with 500 ng RNA sample in each. Next, the two RT replicates were combined to obtain 1 μg cDNA in a total volume of 100 μl (final concentration: 10 ng/μl). This was subsequently diluted to 0.715 ng/μl and 10 μl loaded into each Scorecard well. All Ct values were mapped to their corresponding genes using the TaqMan hPSC Scorecard analysis software provided by the manufacturer. Genes with Ct values < 15 were excluded from further analyses. To be considered for downstream processing, genes were also required to have Ct values < 30 in at least two out of the eight samples. Next, Ct values were linearized (antilog) under the assumption of 100% primer amplification efficiency. The geometric expression mean of the control gene assays was used for subsequent normalization of individual gene expression values. All qPCR data were acquired on a Quant Studio™ 5 Real-Time PCR System (Thermo Fisher Scientific). The thermocycling conditions (SYBR Green reactions) were as follows (ramp rate 1.6C/s for all): 50 degrees Celsius for 2 min, 95 degrees Celsius for 10 min, 40 cycles at 95 degrees Celsius for 15 sec and 60 degrees Celsius for 1 min, followed by melt curve analysis (95 degrees Celsius for 15 sec, 60 degrees Celsius for 1 min, and 95 degrees Celsius for 15 min with ramp rate 0.075 degrees Celsius/sec). The TaqMan hPSC Scorecard thermocycling conditions were as specified by the manufacturer in the accompanying template.
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