RNA-seq of Mus musculus: ventral prostate organoid
Library preparation for RNA sequencing was performed following the high-throughput protocol described in Mildrum et al. J Biomol Tech (2020) with the following modifications: RNA quantification and quality control (QC) were assessed individually using the AATI Fragment Analyzer. 5 replicates of RNA per each sample type were subjected to RNA-seq with QC. Following the template switching reaction, barcoded cDNA samples were pooled, treated with Exonuclease I and cleaned using RNA Ampure beads. Second strand synthesis and PCR amplification were performed using the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer (IDT). 12 cycles of PCR were performed, followed by clean up using regular SPRI beads. Successful amplification of cDNA was confirmed using the Fragment Analyzer. Illumina libraries were then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer. Finally, libraries were cleaned using SPRI beads at 1X and quantified using the Fragment Analyzer and qPCR before being loaded for 40X40 paired-end sequencing using the Illumina NextSeq500.