Rnadvance tissue kit

Samples were kept frozen during the entire procedure preceding RNA extraction using dry ice and liquid nitrogen to flash freeze. Samples were pulverized using Cp02 cryoPREP Automated Dry Pulverizer (Covaris, 500001, Covaris, Woburn, MA) for SVV- and CVA21-treated tumor samples. For SVV samples, 10 mg of pulverized sample was weighed and transferred to a 2 mL microcentrifuge tube (Sample Tube RB, QIAGEN 990381, Hilden, Germany). Buffer RLT Plus with B-mercaptoethanol (600 µL) was added to each sample and lysed. The remaining steps were performed using the QIAcube (QIAGEN, Hilden, Germany), following the manufacturer’s protocol for QIAGEN RNeasy Plus Mini Kit (QIAGEN 74134) under the section for Purification of Total RNA from Animal Tissues. RNA samples were treated with DNAseI (RNAse-free) (New England Biolabs, #M0303S, Ipswich, MA) after extraction.
For CVA21 samples, 10 mg of pulverized sample was weighed and transferred to a 1.5 mL microcentrifuge tube. Lysis Buffer/Proteinase K mixture (400 µL) (RNAdvance Tissue Kit, Beckman Coulter, A32649, Pasadena, CA) was added to each sample. Samples were incubated at 37 °C for at least 30 min to lyse samples completely. The remaining steps were performed using the Biomek i5 (Beckman Coulter, B87583, Pasadena, CA) following manufacturer’s protocol (RNAdvance Tissue Kit). The RNAdvance Tissue Kit includes a DNase I treatment step.

Total RNA was extracted from each tissue sample separately using RNAdvance tissue kit (Beckman Coulter Inc.). We quantified and assessed RNA integrity using a 2100 Bioanalyzer total RNA nano assay (Agilent Technologies). All samples had RNA integrity values greater than 8.0. Libraries were generated using the Illumina TruSeq RNA sample preparation kit v2 (low-throughput protocol) according to the manufacturer’s instructions (Illumina, San Diego, CA). Briefly, 0.4−1.2 µg of RNA was subjected to mRNA selection using poly-T oligo-attached magnetic beads followed by chemical fragmentation (6 min, 94°). The cleaved RNA fragments were then copied into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and Illumina proprietary random hexamer primers. After second-strand synthesis using Illumina supplied consumables, the cDNA was amplified with reagents of the same kit and ligated to barcoded adapters. The final libraries were amplified using 14 PCR cycles. We quantified and assessed library quality on a Bioanalyzer 2100 and randomly pooled equimolar samples in four lanes of the Illumina flowcell (eight or nine samples per lane).