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R N A S E Q U I N S L A B O R ATO R Y P R OTO C O L I N ST R U C T I O N S FO R A D D I T I O N O F S E Q U I N S TO R N A S A M P L E S (for use with RNA sequins version 2) RNA Sequins are designed, validated and manufactured at the Garvan Institute of Medical Research, Sydney Australia. For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. Revision History | Publication Number 1.1.4 | Revision Date November 2016 Material safety data sheets (MSDSs) are available at www.sequin.xyz/downloads/ © Garvan Institute of Medical Research, 2016 TRADEMARKS The trademarks mentioned herein are the property of their respective owners. Qubit® is a registered trademark of Thermo Fisher Scientific and its subsidiaries. Agilent and Bioanalyzer are registered trademarks of Agilent Technologies, Inc. Illumina® is a registered trademark of Illumina, Inc. Clean & Concentrator is a trademark of Zymo Research Corp., Irvine, CA. R N A S E Q U I N S L A B O R ATO R Y P R OTO C O L 1 | INTRODUCTION RNA sequins (RnaQuins) comprise a set of synthetic reference RNA standards that can be added to an RNA sample. They can be used in conjunction with a range of RNA sequencing applications, including spliced-read alignment, isoform assembly, gene discovery and quantitative gene expression profiling. This protocol describes the use of RnaQuin in the laboratory. This includes a step-by-step guide to diluting and adding RnaQuins to your RNA sample prior to library preparation for next-generation sequencing. For a detailed background on the design, validation and use of RnaQuins, please see: ‘Spliced synthetic genes as internal controls in RNA sequencing experiments’ by Hardwick et al., (2016) Nature Methods DOI: 10.1038/nmeth.3958 Additional information on the design of sequins is also available at: www.sequin.xyz/about/transcriptome/ Software, tutorials and other useful informtion on how to analyse sequins is available at: www.sequin.xyz/analysis/ 2 | PREPARATION 2.1 | CONSUMABLES AND EQUIPMENT REQUIRED. Check to ensure that you have all necessary consumables and equipment before thawing your RnaQuin aliquot. See Section 5.1 for related materials and equipment. Table 1. User-Supplied Consumables and Equipment. ITEM Single channel pipettes (10 μl, 20 μl, 200 μl and 1000 μl) Barrier RNase/DNase-free pipette tips Non-binding RNase-free Microfuge Tubes Microcentrifuge Vortex mixer 2.2 | KIT CONTENTS. IMPORTANT | Upon receipt of RnaQuins, check to ensure the mixtures have not been thawed during shipment. Please contact us if there are any concerns with the shipment (sequin@garvan.org.au). For each batch of sequins we manufacture, we perform a number of quality control procedures, including running BioAnalyzer analysis and sequencing a neat mixture randomly selected from each manufactured batch. These quality control resources (including .FASTQ library files) can be downloaded from www.sequin.xyz for quality control and troubleshooting purposes. 2.3 | STORAGE. RnaQuins should always be stored frozen at -80°C and should not be stored in a -20°C frost-free freezer. Each tube contains RNA sequins in 10 μl solution, from which smaller single-use aliquots should be prepared on first thaw to minimize subsequent freeze-thaw cycles. Spin the tube down to collect contents at the bottom of tube prior to aliquoting. PAG E 2 R N A S E Q U I N S L A B O R ATO R Y P R OTO C O L RNA-QUIN V2 Mixture A K562 HUMAN CELLS + Mixture A RNA-QUIN V2 Mixture B GM12878 HUMAN CELLS + Mixture B Figure 1. Example traces of RnaQuins using an 2100 BioAnalyzer with the RNA Nano Kit (Agilent Technologies) for (left upper) neat RnaQuin Mixture A and (left lower) neat RnaQuin Mixture B. Also shown are example traces for (right upper) K562 RNA with Mixture A and (right lower) GM12878 with Mixture B. 3 | WORKFLOW 3.1 | DILUTE RNA SEQUINS STOCK RnaQuins should be ideally added to total RNA samples prior to sample processing steps (such as poly(A) selection or rRNA depletion). Nevertheless, RnaQuins can be added to RNA samples following selection or depletion, but will only provide information on processes downstream to this selection. OPTIONAL | RnaQuins are provided in two mixture fomulations - Mix A and B that contain the same RnaQuin transcripts, but at different molar ratios, thereby emulating fold-change differences in gene expression and alternative splicing between the two mixtures. You can retrieve the mixture files that indicate the concentration of each RnaQuin within both Mix A and Mix B from www.sequin.xyz/downloads. If you are performing a gene-profiling RNAseq experiment to identify differences in gene expression and splicing between two conditions, we suggest that Mix A and Mix B are alternatively a

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