Ion 540 kit chef

Total RNA was isolated from the VOR in vitro q24h multidose simulation study cell pellets (described above) using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol with modifications described above. RNA samples were quantitated using the Qubit RNA high-sensitivity (HS) assay kit and the Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA). RNA (30 ng) was converted to cDNA using the SuperScript Vilo kit (Life Technologies, Carlsbad, CA) per the manufacturer’s protocol. Library barcoding, preparation, and sequencing were carried out per the manufacturer’s protocols using the following Ion Torrent kits on the Ion Chef Prep station and the Ion Torrent S5-XL (Life Technologies, Carlsbad, CA): Ion AmpliSeq transcriptome human gene expression panel Chef-ready kit (catalog no. A31446), Ion 540 chip kit (catalog no. A27766) and Ion 540 Kit-Chef (catalog no. A30011) (Life Technologies, Carlsbad, CA). Eight samples per 540 chip were sequenced with 8 to 10 million reads/sample. Sequencing from the AmpliSeq analysis was analyzed using Partek Flow software to evaluate genes modulated with RPM of >70, an FDR of <0.1, and a VOR/DMSO ratio of >1.5 or <0.75 for each time point.

The AgriSeq targeted-GBS solution utilizes a highly efficient multiplexed PCR chemistry where hundreds to thousands of markers can be targeted and uniformly amplified in a single reaction. Three eighty-four samples were prepared for sequencing using the AgriSeq HTS Library Kit (A34143-Life Technologies). In short, DNA concentrations were normalized to 3.3 ng/µL for a total of 10 ng DNA per 10 µL reaction. Normalized DNA was combined with the AgriSeq custom primer panel and AgriSeq amplification master mix. For amplification of genomic targets, the following thermocycling programs were used; 99°C for 2 minutes, then 15 cycles of 99°C for 15s and 60°C for 4 minutes. Amplicons were prepared for ligation with pre-ligation enzyme digestion at 50°C for 10 minutes, 55°C for 10 minutes, and 60°C for 20 minutes. IonCode™ Barcode Adapters 385-768 Kit (A36546-Life Technologies) were ligated to the digested products with barcoding enzyme and buffer. Labeled amplicons were then pooled, cleaned up, amplified, and normalized. Following library preparation, libraries were loaded onto an Ion 540™ sequencing Chip Kit (A42849) via the Ion 540™ Kit-Chef (A43541-Life Technologies) and Ion Chef. Sequencing was performed on the Ion S5 system (Thermo Fisher, Inc. Waltham, MA). After sequencing, genotyping was performed automatically by Torrent Variant Caller (TVC) on the Torrent Suite Server (TS).

Genomic DNA was extracted on the QIAcube® platform using the QIAamp DNA FFPE tissue kit (Qiagen) according to the manufacturer’s instructions. All DNA samples were then quantified by a Qubit Fluorometer (Termofisher Scientific, Waltham, Massachusetts, USA) using a Qubit® dsDNA HS Assay Kit. Library preparation was performed on 10 ng DNA by the Ion AmpliSeq Library Kit 2.0 (Termofisher Scientific) and the Colon and Lung Panel (Life Technologies) was used to sequencing the entire coding regions of TP53, as previously described [16 (link)]. Briefly, the prepared libraries were sequenced on Ion S5 Sequencer using an Ion 540 Chip and an Ion 540 kit–Chef (all Thermo Fisher Scientific) with configuration 500 flows covering the 200 bp library read length. Raw data were analyzed using the Torrent Mapping Alignment Program aligner implemented in v5.2 of the Torrent Suite software (Thermo Fisher Scientific). All NGS variants were manually reviewed with Integrative Genomics Viewer (IGV version 2.2, Broad Institute, Cambridge, MA, USA) and Biomedical Genomics Workbench Version 4.0 (Qiagen), and then matched against the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and COSMIC (https://cancer.sanger.ac.uk/cosmic) databases.

From liver RNA, libraries were generated using the Ion AmpliSeq™ Transcriptome Mouse Gene Expression Core Panel and Chef-Ready Kit (Comprehensive evaluation of AmpliSeq transcriptome, a whole transcriptome RNA sequencing methodology) (Thermo Fisher Scientific, Waltham, MA). Template-Positive Ion Sphere™ Particles (Thermo Fisher Scientific, Waltham, MA) were loaded on Ion 540™ Chips, using the Ion 540™ Kit-Chef (Thermo Fisher Scientific, Waltham, MA). Sequencing was performed on an Ion S5™ Sequencer with Torrent Suite™ Software v6 (Thermo Fisher Scientific, Waltham, MA). The analyses were performed as previously described^{25 (link)}.

DNA was extracted from 10 μm thick slices of unbaked FFPE tissue using QIAGEN QIAamp DNA FFPE Tissue kits and measured using Qubit 3.0 Fluorometer (Thermo Fischer Scientific). Using 20 ng of DNA, the library was prepared following manufacturers’ instructions. Once the libraries were generated, concentration was measured by quantitative PCR (qPCR) using the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). Following qPCR, the libraries were calculated and pooled together at equal 50 pM concentration for templating on the Ion Chef using the Ion 540 Kit-Chef (2 sequencing runs per initialization; Thermo Fisher Scientific). The samples were then sequenced on the Ion GeneStudio S5 System. Ion Reporter Software was used for mutation load and variant profiling analysis.

Whole genome sequencing was performed using Ion S5 technology, following the previously published procedure [55 (link)]. Briefly, LSDV-positive DNA (~100 ng) from two samples from Lesotho was enzymatically fragmented into 200 bp lengths using Ion shear Plus reagents, then adapters and barcodes were ligated using the Ion Xpress™ Plus Fragment Library Kit and the Ion Xpress barcode adapters (Thermo Fisher Scientific, Berlin, NH, USA). Following size selection using Pippin Prep (Sage Science, Inc., Beverly, MA, USA), the libraries were amplified for eight cycles using Platinum™ PCR SuperMix high-fidelity and library amplification primer mix supplied with the Ion Xpress™ Plus Fragment Library Kit. Equimolar amounts of the barcoded libraries were pooled (100 pM) for automated template preparation using the Ion 540™ Kit-Chef (Thermo Fisher Scientific, USA) and chip leading with the Ion Chef™ Instrument (Thermo Fisher Scientific, USA). Sequencing was performed with 500 flows which generated 200 bp reads on an Ion S5™ Next-generation sequencing system (Thermo Fisher Scientific, USA).

Approximately 100 ng of DNA was enzymatically fragmented into 200 bp lengths using Ion shear Plus reagents. The fragmented DNA was ligated to adapters and barcodes using the Ion Xpress™ Plus Fragment Library Kit and the Ion Xpress barcode adapters (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Size selection was performed using Pippin Prep (Sage Science, Inc., USA). The libraries were further amplified for eight cycles using Platinum™ PCR SuperMix high fidelity and library amplification primer mix provided with the Ion Plus Fragment Library Kit. The amplified barcoded libraries at a concentration of 100 pM were pooled in equal volumes and loaded onto the Ion Chef™ Instrument (Thermo Fisher Scientific, USA) for automated template preparation and chip loading using Ion 540™ Kit-Chef (Thermo Fisher Scientific, USA). Using the Ion Chef™ Instrument, the pooled libraries were clonally amplified on the Ion spheres (ISPs) by emulsion PCR, followed by automated loading template-enriched ISPs onto an Ion 540 chip. Sequencing was performed on an Ion S5™ Next generation sequencing system (Thermo Fisher Scientific, USA) with 500 flows to generate 200 bp reads.