GSE134076
High-depth RNA sequencing of isogenic wild-type, PIK3CA-WT/H1047R and PIK3CA-H1047R/H1047R human iPSCs
We used CRISPR/Cas9 to knock in the cancer "hotspot" mutation PIK3CA-H1047R into one or both alleles of a wild-type induced pluripotent stem cell (iPSC) line (WTC11; Coriell # GM25256; P37-P38). Four cultures from each genotype (3 wild-type clones, 3 heterozygous clones, 2 homozygous clones) were subjected to paired-end mRNA sequencing (mean read length = 150 bp). The aim of this experiment was to confirm and expand upon previous transcriptomic results suggesting a near-binary transcriptional effect in homozygous versus heterozygous PIK3CA-H1047R iPSCs (publication doi: 10.1073/pnas.1821093116).  According to the high-depth transcriptomic dataset, PIK3CA-H1047R/H1047R iPSCs exhibited altered expression of 5644 genes, whereas heterozygous hPSCs showed 492 differentially-expressed genes, supporting a nearly deterministic phenotypic effect of homozygosity for PIK3CA-H1047R.  The differential gene expression analyses were performed based on the limma/voom/eBayes framework (doi: 10.1093/nar/gkv007), using customised scripts with FDR < 0.05 and absolute log2(fold-change) cut-off >= 1.3. Overall design: 4 cultures per genotype (WT/WT, WT/H1047R, H1047R/H1047R), from 3 independent wild-type clones, 3 independent heterozygous clones and 2 independent homozygous clones. All cultures were collected 3 hours following replenishment with growth factor-replete Essential 8/Flex maintenance medium. Moreover, culture collection was conducted over three days according to a block design, thus allowing us to determine transcriptional differences that are robust to biological variability. The library was prepared by The Genomics and Transcriptomics Core (GTC) at the MRC Metabolic Disease Unit, University of Cambridge, using an Illumina TruSeq Standard mRNA Library Prep Kit. The barcoded libraries were pooled and sequenced across 3 lanes on an Illumina HiSeq 4000, with an average depth of approximately 70 million reads per sample.