Quant-iT™ RNA Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Quantitation Range is 5-100 ng	Prepare the Quant-iT™ RNA working solution by diluting the Quant-iT™ RNA reagent 1:200 in Quant-iT™ buffer. Incubate the tubes for 2 minutes at room temperature.

Upstream tips
Quantitation Range is 5-100 ng
Protocol tips
Prepare the Quant-iT™ RNA working solution by diluting the Quant-iT™ RNA reagent 1:200 in Quant-iT™ buffer. Incubate the tubes for 2 minutes at room temperature.

Publication protocol

All RNA samples were DNase-treated with TURBO DNAfree kit (Invitrogen) as per manufacturer’s instructions; RNA integrity and concentration were assessed using Agilent Eukaryotic Total RNA 6000 assay (Agilent Technologies) and Quant-iT™ RNA assay kit on a Qubit™ Fluorometer (Life Technologies Corporation). Given the dynamic nature of genomic technologies, multiple overlapping methods were tested. However, results for tumors within a patient (Tables S1-S3) are consistent with one methodology: NuGen Ovation V2 for MEL38 and MEL218, Illumina TruSeq Stranded for MEL21. The MicroPoly(A)Purist™ Kit (Ambion) was used to enrich for poly(A) RNA from MEL218 and MEL38 DNAse-treated total RNA; MEL21 RNA was ribo-depleted using the Ribo-Zero™ Magnetic Gold Kit (EpiCentre, Madison WI) following the manufacturer protocol. We used either the Ovation® RNA-Seq System V2 (NuGen, 20 ng of either total or polyA RNA), or the Ovation® RNA-Seq FFPE System (NuGen, 150 ng of DNase-treated total RNA) or the TruSeq Stranded Total RNA Sample Prep kit (Illumina, 20 ng ribosomal RNA-depleted total RNA) for cDNA 3 synthesis. All NuGen cDNA sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® with minor modifications. All NuGEN generated cDNA was processed as described previously (9). Briefly, 500 ng of cDNA was fragmented, end-repaired, and adapter-ligated using IDT synthesized “dual same index” adapters. The TruSeq stranded cDNA was also end-repaired and adapter-ligated using IDT synthesized “dual same index” adapters. These indexed adapters, similar to Illumina TruSeq HT adapters, contain the same 8 bp index on both strands of the adapter. Binning reads requires 100% identity from the forward and reverse indexes to minimize sample crosstalk in pooling strategies. Each library ligation reaction was PCR-optimized using the Eppendorf Epigradient S qPCR instrument, and PCR-amplified for limited cycle numbers based on the Ct value in the optimization step. Libraries were assessed for concentration using the Quant-iT™ dsDNA HS Assay (Life Technologies) and for size using the BioAnalyzer 2100 and the Agilent DNA 1000 Assay (Agilent Technologies). The Illumina-ready libraries were enriched using the Nimblegen SeqCap EZ Human Exome Library v3.0 reagent. The targeted genomic regions in this kit cover 63.5 Mb or 2.1% of the human reference genome, including 98.8% of coding regions, 23.1% of untranslated regions (UTRs), and 55.5% of miRNA bases (as annotated by Ensembl version 73 (33)). Each hybridization reaction was incubated at 47º C for 72 hours, and single-stranded capture libraries were recovered and PCR-amplified per the manufacturer’s protocol. Post-capture library pools were sized and mixed at a 1:0.6 sample:AmpureXP magnetic bead ratio to remove residual primer-dimers and to enrich for a library fragment distribution between 300 and 500bp. The pooled capture libraries were diluted to 2 nM for Illumina sequencing. For cDNA-capture data were aligned with Tophat v2.0.8 (params: --bowtie-version=2.1.0 for Ovation; --library-type fr-firststrand -- bowtie-version=2.1.0 for Truseq). For Ovation data, prior to alignment, paired 2x100 bp sequence reads were trimmed with flexbar v 2.21 (params: --adapter CTTTGTGTTTGA - -adapter-trim-end LEFT --nono-length-dist --threads 4 --adapter-min-overlap 7 --maxuncalled 150 --min-readlength 25) to remove single primer isothermal amplification adapter sequences. Expression levels (FPKM) were calculated with Cufflinks v2.0.2 (params--max-bundle-length=10000000--num-threads 4). A visual review step of cDNA capture data was performed to evaluate for expression of MM identified by exome data. 4 Both cDNA-capture (Alt-read number) and FPKM values were considered for candidate prioritization.

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Manufacturer protocol

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