GSM8514795: T1D-01; Homo sapiens; RNA-Seq
Total RNA was isolated from whole blood collected in Tempus® Blood RNA Tubes (ThermoFisher, catalog #4342792). RNA isolation was performed using the Tempus® Spin RNA Isolation Kit (ThermoFisher, catalog t#4380204) according to the manufacturer's instructions. RNA was first evaluated for its quantity and quality using Qubit and Agilent Bioanalyzer; an RNA Integrity Number (RIN) of 7 or higher was used for downstream applications. Five hundred nanograms of total RNA were used for library preparation. For the training cohort, the cDNA library preparation included mRNA enrichment/purification, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification using TruSeq Stranded mRNA Sample Preparation Guide (Illumina, Inc, RS-122-9004DOC, Part #15031047 Rev. E).  Each resulting indexed library with average insert size of 150-200b was quantified, and its quality was assessed by Qubit and Agilent Bioanalyzer, and multiple libraries were pooled in equal molarity. Five microliters of 2 nM pooled libraries per lane were then denatured, neutralized, and applied to the cBot for flow cell deposition and cluster amplification before loading to HiSeq 4000 for 75b paired-end read sequencing (Illumina, Inc.).