Stranded rna seq kit with riboerase

RNA was isolated using procedures common to our lab for next-generation RNA sequencing (Schroder et al., 2015 (link); Hodge et al., 2015 (link); Terry et al., 2018 (link)). Briefly, muscle powder was homogenized in TRIzol (Invitrogen) according to the manufacturer’s directions, then isolated using QIAGEN RNeasy Mini Kit. RNA samples were treated with TURBO DNase (Ambion) to remove genomic DNA. RNA integrity numbers (RIN) were determined using the Agilent 2100 Bioanalyzer. RNA samples with RIN values >8 were used for sequencing library preparation. Then, 250 ng of total RNA was used to prepare barcoded RNAseq libraries using Stranded RNA-Seq Kit with RiboErase (KAPA Biosystems). Samples were sequenced on the HiSeq 2500 (Figure 2) or NovaSeq 6000 (Figure 6; Illumina) using a 2 × 100 kit to a read depth >45 million reads/sample. Reads were checked using FastQC before mapped to mm10 with HISAT2 (Kim et al., 2015 (link)). Gene expression data were collected using HTSeq and DESeq2 analysis packages (Love et al., 2014 (link)), and splicing analysis was performed using the protocol outlined by Schafer et al., 2015 (link). Exon usage data is reported as PSI and change in PSI (∆PSI; iMSBmal1^-/- - iMSBmal1^+/+). Data have been deposited in NCBI’s Gene Expression Omnibus and are accessible using the GEO accession number GSE189865.