INSTRUCTIONS FOR ADDITION OF SEQUINS TO RNA SAMPLES (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.
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. PAGE 2
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 added to seperate samples from each condition being compared (ensure that you do not add both mixtures to a single sample). This enables the use of RnaQuins in a subsequent assessment of the fold-change differences identified between conditions. RnaQuins are provided in solution in nuclease-free water at a concentration of 15.0 ng/μl, which is sufficient for more than 100 NGS sample library preparations. However, the amount of RNA sequins to add to your sample RNA depends on the library preparation technique used: Table 2. Guidelines for diluting RnaQuin Standard Mixes according to sample RNA amounts / library preparation method. RNAQUIN MIX A or B (dilution) SAMPLE RNA AMOUNT TOTAL RNA POLY-A RNA 20 ng 4 μl (1:2,500) 2 μl (1:25) 50 ng 1μL (1:250) 5 μl (1:25) 100 ng 2 μl (1:250) 1 μl (1:5) 500 ng 1 μl (1:25) 2 μl (neat) 1000 ng 2 μl (1:25) - 5000 ng 1 μl (1:5) - PAGE 3
OPTIONAL The above protocol adds RNA sequins at a relative fraction according to the amount of RNA in the sample. However, RNA sequins can alternatively be added at an absolute amount, relative to the RNA sample amount. For example, 1ng of RNA sequins may be added to multiple samples, regardless of differences in RNA amount. This can be particularly useful when the total RNA population size changes between samples, and enables absolute measurements of gene expression and foldchanges between samples that avoids confounding artefacts of normalisation using library size. For further information, please refer to - Owens et. al., Measuring Absolute RNA Copy Numbers at High Temporal Resolution Reveals Transcriptome Kinetics in Development. (2016) Cell Reports Jan 26;14(3):632-47 A. Total RNA (that has not yet undergone poly(a) selection or rrna depletion) Table 2 provides guidelines for the amount of RnaQuins to add depending on the starting amount of total RNA that will be used in the selection/depletion procedure. For example, if the starting amount of total RNA for the selection/depletion procedure is 1 μg, add 2 μl of a 1:25 dilution of RnaQuin Standard Mix A or Mix B. B. RNA that has undergone poly(a) selection Table 2 provides guidelines for the amount of RnaQuins to be added to mrna that has been purified following by poly-a selection. C. rrna-depleted RNA, Table 2 provides guidelines for the amount of RnaQuins to add depending on the amount of total RNA that was initally used in the previous rrna-depletion steps. For example, if the rrna-depletion procedure used 1 μg of total RNA, add 2 μl of 1:25 dilution of RnaQuin Standard Mix A or Mix B to the total amount of rrna- depleted RNA recovered. These guidelines for adding RnaQuin Standard Mixes to RNA samples have been developed and validated by sequencing library preparation, Agilent 2100 Bioanalyzer assessment, and bioinformatic sequence analysis. However, the amount of library reads assigned to RnaQuins is directly proportional to their dilution in the RNA sample and this dilution can be adjusted and modified by the end user depending on their final use of RNA sequins. 3.2 ADDITION OF RNA SEQUINS TO RNA SAMPLES. We recommend users prepare a fresh dilution of the RnaQuin sequins from single-use aliquots (see Section 2.3) Prepare the appropriate dilution required of each RnaQuin Standard Mix according to Table 3. If more than 5 ul or 10 μl of the dilution are needed, scale the volumes accordingly and add the volume of the diluted RnaQuin Mix to each RNA sample. Table 3. Guidelines for preparing RnaQuin Mixture dilutions. DILUTION STANDARD MIX NUCLEASE-FREE WATER 1:5 1 μl undiluted 4 μl 1:25 1 μl of 1:5 4 μl 1:250 1 μl of 1:25 9 μl 1:2,500 1 μl of 1:250 9 μl Add only one freshly diluted RNA sequin mixture (either mixture A or mixture B) preparation to each single RNA sample. PAGE 4
3.3 DOWNSTREAM LIBRARY PREPARATION WITH SEQUIN-CONTAINING RNA SAMPLES. Use the combined sample as input according to your preferred library preparation kit s protocol. OPTIONAL Downstream library preparation workflow may require user s to concentrate the sample RNA after the addition of the RNA sequins. Sequin-containing RNA samples can be concentrated by either ethanol precipitation, SPRI bead purification (e.g. RNAClean XP, Beckman Coulter), column-based methods (e.g. RNA Clean & Concentrator Kit, Zymo Research), or using vacuum centrifugation (e.g. Eppendorf Vacuum Concentrator Plus). K562 + MIXTURE A GM12878 + MIXTURE B Figure 2. Successful RnaQuin-containing Total RNA Libraries. A, K562 with Mix A. B, GM12878 with Mix B. Samples analysed by Agilent 2100 BioAnalyzer trace (size distributions sequenced on an Illumina HiSeq 2500 Instrument). 4 EXAMPLE WORKFLOW Here we describe an example workflow to add the RNA sequin mixture A to total RNA harvested from the K562 human cell-line for sequencing on the Illumina HiSeq 2500 Instrument. 4.1 For whole-transcriptome analysis using Illumina System next-generation sequencing, 8 μl of a 1:25 dilution of RnaQuin Mixture A was added to 4 μg of Total RNA from K562 cell-type (Coriell Institute for Medical Research GM05372). 4.2 Following the addition of RnaQuin Mix A to the K562 Total RNA, the RnaQuin Mix-containing total RNA mixture was used as input Total RNA in Step 2.7 (page 10) of the KAPA Biosystems protocol for the KAPA Stranded mrna-seq Kit (Illumina platform KR0960 v2.14). 4.3 Subsequent steps were performed as per manufacturer s protocol, until step 12.14 (page 14), where 22 μl of nucleasefree water was used to liberate the DNA from the Agencourt AMPure XP beads. 4.4 Additionally, at step 12.17 (page 14) the 20 μl supernatant was carefully transferred to a 1.5 ml Lo-Bind microcentrifuge tube (Eppendorf). 4.5 The purified cdna fragment library was quantified using the BR dsdna Qubit Assay on a Qubit 3.0 Fluorometer (Life Technologies) and verified on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit (Agilent Technologies) (see Figure 2). PAGE 5
5 SUPPLEMENTARY INFORMATION 5.1 RELATED PRODUCTS. Description and catalogue accession of products that can be used in conjunction with RnaQuins. Table 4. Related products referred to in guide. PRODUCT PART NUMBER (SOURCE) Agencourt RNAClean XP A63987 (Beckman Coulter) Agilent RNA 6000 Nano Kit 5067-1511 (Agilent Technologies) Agilent High Sensitivity DNA Kit 5067-4626 (Agilent Technologies) Epicentre Ribo-Zero Magnetic Kit MRZH11124 (Epicentre Biotechnologies) Qubit RNA HS/BR Assay Kit Q32852; Q10210 (Life Technologies) Qubit DNA HS/BR Assay Kit Q32854; Q32850 (Life Technologies) KAPA Stranded mrna-seq Kit KK8420 (KAPA Biosystems) RNA Clean & Concentrator -5 kit R1015 (Zymo Research) Eppendorf Vacuum Concentrator Plus 5305 000.380 (Eppendorf) 6 TECHNICAL ASSISTANCE For technical assistance, contact: Website Email www.sequin.xyz sequin@garvan.org.au PAGE 6