RNA was collected from T1 and T2 cell cultures polarized for 5 days using TRIreagent and complementary DNA synthesized using SuperScript First Strand Synthesis (ThermoFisher). Quantitative real time PCR measured expressed RNAs using SYBR green master mix (Applied Biosystems). RNA levels were standardized relative to the housekeeping gene GAPDH. Patient samples containing definite outliers (ROUT with Q=0.1% by GraphPad Prism software) were removed. Patient samples with both T1 and T2 available were included for analysis.
For RNA sequencing, RNA was collected by TRIreagent followed by DNase digestion. Library preparation was performed with the Illumina Tru-Seq Stranded RNA kit. RNA sequencing was performed with an Illumina HiSeq2500 instrument by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facilities consecutively on all samples. 100bp paired end reads were generated. Average sequencing depth of all samples was 43 million mapped reads ± 13.5 million (standard deviation). FASTQ files were processed using DESeq2 to identify differentially expressed genes (33 (link)). Data are available in NCBI’s Gene Expression Omnibus (34 (link)) with GEO series accession number GSE185193. GO Enrichment analysis for overrepresented biological processes was performed using the Gene Ontogeny Resource (35 (link)–37 (link)).
For RNA sequencing, RNA was collected by TRIreagent followed by DNase digestion. Library preparation was performed with the Illumina Tru-Seq Stranded RNA kit. RNA sequencing was performed with an Illumina HiSeq2500 instrument by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facilities consecutively on all samples. 100bp paired end reads were generated. Average sequencing depth of all samples was 43 million mapped reads ± 13.5 million (standard deviation). FASTQ files were processed using DESeq2 to identify differentially expressed genes (33 (link)). Data are available in NCBI’s Gene Expression Omnibus (34 (link)) with GEO series accession number GSE185193. GO Enrichment analysis for overrepresented biological processes was performed using the Gene Ontogeny Resource (35 (link)–37 (link)).
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