Agilent rna 6000 nano kit on 2100 bioanalyzer

The viral RNA extraction was performed using QIAamp® DSP Virus Kit (Qiagen, Hilden, Germany). The protocol was performed as recommended by the manufacturer with one additional step, the treatment with RNase-Free DNase Set (QIAGEN). The quality of the extracted RNA was evaluated with Agilent RNA 6000 Nano Kit on 2100 Bioanalyzer and low contamination with ribosomal RNA (rRNA) was observed. Viral extraction was followed by DNA library preparation that was conducted with TruSeq Stranded Total RNA kit (Illumina, San Diego, CA, USA) without the rRNA depletion procedure. Quantification of DNA libraries was done using Qubit™ dsDNA HS Assay Kit and the Qubit4 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States). The quality of the DNA libraries was determined by Agilent 2100 Bioanalyzer using High Sensitivity DNA kit. The average size of the libraries was 260 bp. Finally, DNA libraries consisting of 4−5 samples were sequenced using Illumina® MiSeq® Reagent Kit v3 with a number of sequencing cycles equal to 150 and a load concentration of 10 pM per library.

To perform RNA sequencing, corneal epithelial layer was separated and total RNA was extracted from three independent biological samples. rRNA depletion was performed using Ribo-Zero Gold rRNA removal kit H/M/R (Illumina). Agilent RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent) was used to do total RNA sample QC. Libraries were constructed with a series of standard steps, included RNA fragment and reverse transcription to double-strand cDNA, end repair, tailing and adaptor ligation, PCR amplification, denaturation, and cyclization. The final library was single-strand circle DNA (ssCir DNA) and was amplified with phi29 (Thermo Fisher Scientific) to make DNA nanoball (DNB). DNBs were transformed to single-end 50 bases reads and sequenced on BGISEQ-500 platform (BGI-Shenzhen, China).

RNA integrity was assessed with the Agilent RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent) followed by library preparation with the Ion AmpliSeq Transcriptome Human Gene Expression Panel (Thermo) according to the manufacturer’s protocol [117 (link)]. Briefly, equal amounts of total RNA from all samples were reverse transcribed and the cDNAs were subjected to multiplex PCR to amplify parts of the target transcripts. The resultant amplicons were partially digested AND ligated with adapters. Ligation products were then purified with AMPure^® XP beads (Beckman). The library was quantified in the Bioanalyzer 2100 and the concentration was adjusted to ∼100 pM prior for template preparation. Then, eight barcoded library templates were clonally amplified on Ion Sphere particles (ISPs) with the Ion PI IC 200 Kit (Thermo) on the Ion Chef Instrument (Thermo Fisher Scientific, USA), loaded onto Ion PI chips and sequenced on an Ion Proton sequencer (Thermo) with the Ion PI IC 200 Kit, according to the manufacturer’s instructions.