Ribo zero rrna removal kit human mouse rat

RNA purity was determined using a NanoDrop ND1000 spectrophotometer. Total RNA sequencing libraries were prepared according to the manufacturer's instructions using a Truseq stranded total RNA sample prep kit (Illumina, San Diego, CA, USA). One microgram of total RNA was further treated for ribosomal RNA (rRNA) depletion using Ribo-zero rRNA removal kit (human/mouse/rat) (Illumina). Then, the rRNA-depleted total RNA was fragmented into small pieces at 94°C for 8 minutes. The cleaved RNA fragments were copied to first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNase H. Finally, one 'A' base was added, and the adapter was subsequently ligated. The products were purified and enriched using PCR to create the final cDNA library. After real-time PCR, index-tagged libraries were combined in equimolar ratio. RNA sequencing was performed using the Illumina NextSeq 500 platform according to the manufacturer's recommended protocol.
The reads were aligned with the gene expression values using Cufflinks (fragments per kilobase of transcript per million mapped reads). The differentially expressed genes of the vehicle treatment group were compared to that of the PA or mmLDL treatment group using Cuffdiff. Upregulated or downregulated genes with q-value <0.05 and 2-fold change were identified.

Full protocol details were reported in our previous study.6 (link) In brief, sequencing libraries were prepared using a NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, USA) according to the manufacturer’s protocol. Ribosomal (r)RNA was removed from the total RNA using Ribo-Zero™ rRNA Removal kit (Human/Mouse/Rat) (Illumina, San Diego, CA, USA). The libraries were sequenced on an Illumina HiSeq instrument (Illumina), generating 150-bp pair-end reads.