Neb next ultra rna library prep kit

Total RNA was extracted from using TRIzol (Invitrogen) combined with Purelink RNA columns (Fisher) and quantified using a Nano-drop. mRNA was reverse transcribed into cDNA using the ABI High-Capacity cDNA Synthesis kit (ABI). Real-time PCR was performed on an ABI7900HT PCR machine using SYBR green fluorescent dye (Applied Biosystems). Fold changes were calculated using the ΔΔCT method, with Tata Binding Protein (Tbp) mRNA serving as a normalization control. RNaseq libraries were prepared using the NEB Next Ultra RNA Library Prep Kit and sequenced on a NovaSeq 6000 (PE150) (Novogene). RNA-seq reads were aligned to UCSC mm9 genome using STAR aligner (Dobin et al., 2013 (link)) with an option, “–outSAMstrandField intronMotif–outFilterMultimapNmax 1.” Mitochondrial reads were filtered out to avoid sequencing depth bias due to mitochondrial abundance. Then, reads aligned to genes were counted using featureCounts (Liao et al., 2014 (link)). Differential gene expression analysis was performed using edgeR (Robinson et al., 2010 (link)). Hierarchical clustering was performed to identify distinct functional modules of genes using Ward’s criterion and Pearson correlation as a similarity measure. Gene ontology analysis was done using EnrichR (Chen et al., 2013 (link)).