title: CARE Cohort Longitudinal RNA-seq
design: The quality of total RNA samples was first assessed using Agilent 2100 Bioanalyzer and the concentration of the total RNA samples was measured using Thermo Scientific NanoDrop. Based on the concentration obtained from NanoDrop, two hundred nanograms of RNA per sample were used to prepare dual-indexed strand-specific cDNA library using Kapa mRNA HyperPrep kit (KapaBiosystems) along with QIAseq FastSelect Human Globin removal kit (Qiagen). Libraries were prepared in a 96 well plate using the Biomek FxP Laboratory Automation Workstation. The resulting libraries were assessed for their quantity and size distribution using Agilent 4200 TapeStation. Each plate was pooled using the QIAgility Automation System. Two and a half nanomolar pooled libraries were utilized per lane on a flowcell sequenced with 2×150bp paired-end configuration on NovaSeq6000 (Illumina) using Xp workflow with NovaSeq6000 S4 Reagent kit (300 cycle) and NovaSeq Xp 4-lane manifold kit. A Phred quality score (Q score) was used to measure the quality of sequencing.