title: RNA-Seq of rat prostate stroma
design: Large RNA libraries (non-coding and mRNA) were constructed using the KAPA RNA HyperPrep kit with RiboErase according to the manufacturers instructions with some modifications. NEBNext Multiplex Oligos for Illumina was used for the adaptor and barcodes. Libraries were incubated at 37C for 15 minutes with the USER enzyme (NEB) before the final amplification. qPCR was used to determine cycle number with the KAPA RealTime Library Amplification Kit. Size selection (200-700 bp) was done using KAPA Pure beads. Quality control analysis was done with the Agilent DNA High Sensitivity chip and final concentration was determined with the Qubit dsDNA high sensitivity assay. Pooled libraries were sequenced with paired-end 100 bp sequencing on the Illumina HiSeq 4000 sequencer.Small RNA libraries were constructed with the NEBNext Multiplex Small RNA Library Prep Set for Illumina and were barcoded with NEBNext Multiplex Oligos for Illumina. Purification and size selection was done with the KAPA Pure beads following the protocol. An additional size selection (115-160 bp) was performed using the Pippin Prep 3% gel with marker P (Sage Science). Concentration was determined using the Qubit dsDNA high sensitivity assay (Thermo Fisher) and quality control was done with Agilent DNA High Sensitivity Chip. Libraries were pooled and concentrated using KAPA Pure beads (2.2X), and sequenced with a custom sequencing primer: 5-ACA CGT TCA GAG TTC TAC AGT CCG A-3 on the Illumina HiSeq 4000 sequencer (single-end 50 bp).