Transcriptional Profiling of Differentiated THEKs

RNA-Seq libraries were prepared for transcriptomics analysis as described (102 (link)). THEKs were grown to confluence in 10 cm cell culture dishes in KSFM, then differentiated in E-medium for 72 hours. RNA was isolated from THEKs using the RNeasy Kit (QIAGEN) according to the manufacturer’s instructions. mRNAs were isolated using the NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs). RNA-Seq libraries were prepared for Illumina using the NEBNext Ultra-Directional RNA library preparation kit (New England Biolabs). Library quality was confirmed using an Agilent BioAnalyzer 2100 and quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs). Sequencing was performed using the Illumina NextSeq500 platform employing a single-end, 75-base pair sequencing strategy. All RNA-Seq reads were then aligned to the Homo sapiens reference genome (UCSC hg19, RefSeq and Gencode gene annotations) using RNA STAR under default settings. Fragments per kilobase per million mapped fragments generation and differential expression analysis were done using the DESeq2 package. Statistical significance was determined using an adjusted P value ≤ 0.05.