Ovation solo rna seq library preparation kit

RNA samples containing a minimum of 1 ng RNA and a RIN > 4 were used for further RNAseq analysis. First, RNA was amplified using the Ovation^® SoLo RNA-seq library preparation kit (NuGEN) used for low input or single-cell sequencing according to manufacturer’s instructions, including a ribosomal RNA depletion step (Insert Dependent Adaptor Cleavage). Libraries were then monitored for concentration and fragment sizes using a Fragment Analyzer (kit Standard Sensitivity NGS) and by qPCR (ROCHE Light Cycler 480) prior sequencing on Illumina HiSeq2000 platform (single end 50 bp length).

A total of 5 ng RNA per sample was used as input material for sequencing libraries using the Ovation® SoLo RNA-Seq Library Preparation Kit (NuGEN, CA, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. For small RNA libraries, a total of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. The clustering of the index-coded samples was performed on acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced using an Illumina Hiseq platform and paired-end reads were generated [25 (link), 26 (link)].

RNA was extracted from 300K cells using the Zymo Direct-zol miniprep kit (Cat# R2051) and RNA quality assessed using the Agilent DNA tape screen assay. The RNA Integrity Number (RIN) scores for all samples were > 7.0. Total RNA libraries were generated using the NuGEN Ovation® SoLo RNA-Seq Library Preparation Kit (Cat# 0500–96). Libraries were sequenced using Illumina NovaSeq 6000 instrument with S4 flow cell and 150 base pair paired-end reads.FASTQ files were generated from the NovaSeq BCL outputs and quality was assessed with FASTQC²⁹ . Differentially expressed genes were identified between subjects with different disease severity using the limma package and voom to model variance^{30 (link)}.