TrialNet KQ1 Immune Cell Transcriptomic Study
RNA-seq libraries were prepared in batches of 48 using the SMARTer® Stranded Total RNA-Seq v2 Pico kit (Takara) with 1 ng RNA input, including one external control per batch. Libraries were pooled (n = 96) and sequenced (75 bp paired-end) on Illumina NovaSeq6000 (S4 flow cells, XP protocol) to ~10 M reads/sample. Reads were quality-checked (FastQC), adaptors trimmed (TrimGalore), and rRNA reads removed (BBSplit). Alignments to GRCh38 were performed with HISAT2 (alignment rate >95%). Count matrices were generated with featureCounts (Rsubread) and stored as DGEList objects (edgeR). Lowly expressed genes (<5 CPM) were filtered with filterByExpr. Counts were variance-stabilized (vsn), and genes annotated using the Bioconductor package org.Hs.eg.db; 81 unannotated genes were removed. The 6,000 most variable genes were selected based on median absolute deviation. Sequencing saturation was confirmed with RNA-SeQC. Sample identity was verified with NGSCheckMate, and mismatches removed.
NIDDK-phs004311
TNRNA01373