TailorMix Small RNA Sample Preparation Kit Catalog Numbers: TM100-A and TM100-B Introduction The TailorMix Small RNA Sample Preparation Kit from SeqMatic is a comprehensive solution for generating small RNA libraries for the Illumina sequencing platform, enabling the discovery and profiling of small RNAs from various organisms and tissues. Our unique TailorMix solution has been developed for simplicity and reproducibility without sacrificing quality or yield. This user guide describes the necessary steps to generate libraries for cluster generation and sequencing. The steps for library generation include adapter ligation, cDNA synthesis, PCR amplification, and gel purification (Figure 1). Figure 1 TailorMix Small RNA Sample Preparation Overview Features  User friendly workflow – libraries can be prepared in a single day with under one hour of hands on time.  Comprehensive sample prep kit – most components are supplied as ready-to-use mixtures which improves consistency and reproducibility.  Wide dynamic range – requires as little as 50 ng of Total RNA input. 29528 Union City Blvd. Union City, CA 94587 Tel: 510.870.0965 www.seqmatic.com [email protected] TailorMix Small RNA Sample Preparation Kit Components Each TailorMix Small RNA Sample Preparation Kit contains one set of sample prep reagents (12 samples), one set of 12 unique barcodes, and a set of gel purification tools. The reagents and should be stored at -15 °C to -25 °C. The kit is designed to be stable for up to six months after the shipping date. Set 1: Sample Prep Reagents 1. 2. 3. 4. 5. 6. 7. 8. 9. 3’ Adapter Ligation Mix A 3’ Adapter Ligation Mix B 3’ Adapter Ligation Mix C 5’ Adapter Ligation Mix A 5’ Adapter Ligation Mix B 5’ Adapter Ligation Mix C cDNA Synthesis Mix A cDNA Synthesis Mix B PCR Master Mix Set 2: Barcode Primers Catalog # TM100-A PCR Primer 1 Barcode 1 Barcode 2 Barcode 3 Barcode 4 Barcode 5 Barcode 6 Barcode 7 Barcode 8 Barcode 9 Barcode 10 Barcode 11 Barcode 12 Catalog # TM100-B PCR Primer 1 Barcode 13 Barcode 14 Barcode 15 Barcode 16 Barcode 17 Barcode 18 Barcode 19 Barcode 20 Barcode 21 Barcode 22 Barcode 23 Barcode 24 Set 3: Gel Purification Tools 1. 2. 3. 4. 5. Custom DNA Ladder Gel Cutter Tool Gel Breaker Tool 2.0 ml DNA Collection Tube 5 μm Filter Tubes 2 TailorMix Small RNA Sample Preparation Kit Consumables Preparation The kit contains all necessary reagents to perform the experiment with the exception of common consumables and instruments. Please make sure all equipment is available before starting this experiment (Table 1). Table 1 List of Consumables and Equipment Consumables and Equipment 1.5 ml nuclease-free microcentrifuge tubes 200 μl, clean, nuclease-free PCR tubes Nuclease-free water Supplier General lab supplier General lab supplier General lab supplier Cooler block IST Engineering, 6388-001 Thermal cycler General lab supplier 5X TBE buffer General lab supplier 6% TBE Gels 1.0 mm, 10 Well LIFE Technologies, EC6265BOX Hi-Density TBE Sample Buffer(5X) LIFE Technologies, LC6678 50bp DNA Ladder (Optional) General lab supplier SYBR® Gold Nucleic Acid Gel Stain LIFE Technologies, S-11494 XCell SureLock® Mini-Cell LIFE Technologies, EI0001 Electrophoresis power supply General lab supplier Dark reader transilluminator Bench top microcentrifuge Tube shaker or thermal mixer NanoDrop 2100 Bioanalyzer DNA 1000 chip High Sensitivity DNA chip General lab supplier General lab supplier General lab supplier Thermo Scientific Agilent Technologies Agilent Technologies, 5067‐1504 Agilent Technologies, 5067‐4626 Best Practices       Always wear gloves and use sterile technique. Set up reactions using sterile non-stick nuclease-free tubes. Place samples and reagents on ice at all times and avoid extended pauses. Reagents should be prepared using RNAse-free components to avoid contamination. Prepare an extra 10% mixture when running multiple samples. Avoid repeated freeze/thaw cycles. RNA Input This protocol has been optimized using purified 100 ng of high quality human brain total RNA as input. Because small RNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. You may also use isolated miRNA as starting material. 3 TailorMix Small RNA Sample Preparation Kit Small RNA Library Sample Preparation Protocol 3’ Adapter Ligation Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 28°C if available. 1. Denature the 3’ adapter by assembling the following components in a sterile 200 μl PCR tube: Reagent 100 ng Total RNA 3’ Adapter Mix A Total Volume (μl) 5 2 7 2. Gently pipette mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice. 3. Set up following the 3’ Adapter Ligation reaction: Reagent Denatured 3’ adapter and RNA 3’ Adapter Mix B Total Volume (μl) 7 2 9 4. Gently pipette mix thoroughly and incubate at 28°C for 1 hour and immediately add 2 μl of 3’ Adapter Ligation Mix C directly into each sample tube while remaining on the thermal cycler. 5. Gently pipette mix thoroughly and continue to incubate the tube at 28°C for an additional15 minutes and then place the tube on ice. 5’ Adapter Ligation 6. Aliquot 2 μl of 5’ Adapter Ligation Mix A into a separate sterile, nuclease-free 200μl PCR tube. 7. Incubate the tube at 70°C for 1 minute to and then place the tube on ice. 8. Set up following the 5’ Adapter Ligation reaction: Reagent 3’ Adapter Ligated RNA Denatured 5’ adapter 5’ Adapter Ligation Mix B 5’ Adapter Ligation Mix C Total 9. Volume (μl) 9 2 2 2 15 Gently pipette mix thoroughly and incubate at 28°C for 30 minutes and then place the on ice. 4 TailorMix Small RNA Sample Preparation Kit cDNA Synthesis Pre-heat the thermal cycler to 50°C. 10. Set up following the cDNA Synthesis reaction on a new sterile 200 μl PCR tube on ice. Reagent 3’ and 5’ Adapter Ligated RNA cDNA Synthesis Mix A cDNA Synthesis Mix B Total 11. Volume (μl) 5.5 3.5 2 11 Gently pipette mix thoroughly and incubate at 50°C for 1 hour and then place the tube on ice. PCR Amplification 12. Set up following the PCR reaction: Reagent cDNA PCR Master Mix PCR Primer 1 Barcode Primer* Total Volume (μl) 11 35 2 2 50 *Only one of the barcode primers is used for each reaction. 13. Gently pipette mix thoroughly and amplify the samples in the thermal cycler using the following PCR cycling conditions: - 98°C for 30 seconds - 13 cycles of: - 98°C for 2 seconds - 60°C for 30 seconds - 72°C for 15 seconds - 72°C for 5 minutes - Hold at 4°C PCR yield can be monitored by running an Agilent DNA 1000 assay using a dilution of 1 μl of PCR product and 4 μl of nuclease-free water. If the amount of product is insufficient, the number of PCR cycles can be adjusted up to 15 cycles. Sample Pooling The TailorMix Small RNA Sample Preparation kit is capable of multiplexing up to 24 samples into a single lane of an Illumina flow cell. While processing samples in parallel, incorporate the barcode at the PCR step. Samples can be pooled prior to library purification step. 5 TailorMix Small RNA Sample Preparation Kit Library Purification 14. Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis. 15. Assemble the gel electrophoresis apparatus. 16. Mix 2 μl of custom ladder with 2 μl of DNA loading dye. 17. (Optional) Mix 1 μl of DNA ladder with 1 μl of DNA loading dye. 18. Add 10 μl Hi-Density TBE Sample Buffer to PCR product from step 19 19. Load 2 μl of the mixed custom ladder into two wells of the 6% PAGE gel separated by two wells for the PCR product 20. (Optional) Load 2 μl of the mixed DNA ladder into a separate well. 21. Load 22 μl of the mixed amplified cDNA construct into two wells. 22. Run the gel for 1 hour at 145V and immediately remove the gel from the apparatus. Recover Purified Library 23. Open the gel cassette and stain with Sybr Gold according to the manufacturer’s instructions. 24. Place the gel on a Dark Reader Transilluminator. 25. Insert the gel cutter tool to the end of a P1000 pipette. 26. Align the gel cutter tool between the 160 bp and 140 bp band of the custom ladder on the sample lane and press down firmly into the gel and excise the gel fragment by pipetting up. 27. Pipette the gel fragment into the gel breaker tube. 28. Centrifuge the gel breaker tube in a bench top centrifuge at 20,000 xg for two minutes at room temperature. Ensure that all of the gel has moved through the holes into the collection tube. 29. Add 100 μl of nuclease free water or TE buffer to the collection tube containing the gel debris. 30. Elute the DNA by shaking the tube at room temperature for at least two hours. The tube can be shaken overnight if desired. 31. With a P1000 pipette, transfer the eluate and gel debris into the top of a 5 μm filter tube. 32. Centrifuge the filter tube at 600 xg for 10 seconds. The library is now ready for validation and cluster generation. 6 TailorMix Small RNA Sample Preparation Kit Library Validation Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library. 1. Use 1 μl of resuspended construct on a High Sensitivity DNA chip. 2. Check the size, purity and concentration of the sample. Figure 2 High Sensitivity DNA Chip Trace of the Final Library from an Adult Normal Brain Tissue Total RNA Sample Small RNA Library bp 7