TailorMix Stranded mrna Sample Preparation Kit

Transcription

1 TailorMix Stranded mrna Sample Preparation Kit Catalog Numbers: TM200-A and TM200-B Introduction The TailorMix Stranded mrna Sample Preparation Kit from SeqMatic is a comprehensive solution for generating strand specific RNA libraries for the Illumina sequencing platform. Our kits enable researchers to detect SNPs, alternative splicing, and gene expression with high confidence. The unique TailorMix reagents and workflow have been developed for simplicity and reproducibility without sacrificing quality or yield. Figure 1 TailorMix Stranded 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 Union City Blvd. Union City, CA Tel: info@seqmatic.com

2 Components Each TailorMix Stranded RNA Sample Preparation Kit contains one set of sample prep reagents (12 samples) and one set of 12 unique barcodes. The reagents 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 s 1. mrna Extraction Beads 2. Elution Buffer 3. Mix A 4. Mix B 5. Mix C 6. Mix D 7. Mix E 8. Mix F 9. Mix G 10. Mix H 11. Mix I 12. Mix J 13. Mix K Set 2: Barcode Primers Catalog # TM200-A Barcode Sequence Catalog # TM200-B Barcode Sequence PCR Primer PCR Primer Barcode 1 CGTGAT Barcode 13 TTGACT Barcode 2 ACATCG Barcode 14 GGAACT Barcode 3 GCCTAA Barcode 15 TGACAT Barcode 4 TGGTCA Barcode 16 GGACGG Barcode 5 CACTGT Barcode 17 CTCTAC Barcode 6 ATTGGC Barcode 18 GCGGAC Barcode 7 GATCTG Barcode 19 TTTCAC Barcode 8 TCAAGT Barcode 20 GGCCAC Barcode 9 CTGATC Barcode 21 CGAAAC Barcode 10 AAGCTA Barcode 22 CGTACG Barcode 11 GTAGCC Barcode 23 CCACTC Barcode 12 TACAAG Barcode 24 GCTACC Enabling Accurate NGS 2

3 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 Supplier 1.5 ml nuclease-free micro centrifuge tubes General lab supplier 200 μl, clean, nuclease-free PCR tubes General lab supplier Nuclease-free water General lab supplier Magnetic Pulling Block IST Engineering, RNA Clean & Concentrator-5 Agencourt AMPure XP Zymo Research, R1015 Beckman Coulter, A63881 Cooler block IST Engineering, Thermal cycler General lab supplier Bench top microcentrifuge General lab supplier Tube shaker General lab supplier 2100 Bioanalyzer Agilent Technologies High Sensitivity DNA chip Agilent Technologies, 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. s 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. The use of total RNA from other tissue or species may require optimization. You may also use isolated mrna as the starting material and skip the mrna extraction section of the protocol. Enabling Accurate NGS 3

4 Stranded mrna Library Sample Preparation Protocol mrna Extraction 1. Dilute the total RNA sample to a volume of 50 μl using nuclease free water. Incubate the sample at 75 for 1 minutes and then place the tube on ice. 2. Resuspend the mrna extraction beads by pulse vortexing. 3. Add 50 μl of resuspended RNA extraction beads and pipette mix thoroughly. 4. Place the tube on a 1400rpm shaker at 25 C for 5 minutes. 5. Place the tube on the magnetic stand for 1 minute or until the solution becomes clear. Discard all of the supernatant in the tube. 6. Take the tube off of the magnetic stand and wash the beads using 200 μl of Bead Wash Buffer. Pipette mix thoroughly. 7. Place the tube on the magnetic stand for 1 minute or until the solution becomes clear. Discard all of the supernatant in the tube. 8. Add 50 μl of Elution Buffer and pipette mix thoroughly. 9. Incubate at 75 C for 2 minutes and immediately place the tube on the magnetic stand for 1 minute or until the solution becomes clear. Transfer the supernatant to a new tube. 10. Add 50 μl of resuspended RNA extraction beads and pipette mix thoroughly. 11. Place the tube on a 1400rpm shaker at 25 C for 5 minutes. 12. Place the tube on the magnetic stand for 1 minute or until the solution becomes clear. Discard all of the supernatant in the tube. 13. Take the tube off of the magnetic stand and wash the beads using 200 μl of Bead Wash Buffer. Pipette mix thoroughly. 14. Place the tube on the magnetic stand for 1 minute or until the solution becomes clear. Discard all of the supernatant in the tube. 15. Add 17 μl of nuclease free water and pipette mix thoroughly. 16. Incubate at 75 C for 2 minutes and immediately place the tube on the magnetic stand for 2 minutes or until the solution becomes clear. 17. Transfer the supernatant to a new tube. Enabling Accurate NGS 4

5 RNA Fragmentation 18. Set up the following RNA Fragmentation reaction: mrna from step Mix A 2 Total Incubate at 94 C for 4 minutes and then immediately place the tube on ice for at least 1 minute. Pulse spin the tube to collect any condensation which may have formed on the side walls of the tube. PNK Treatment 20. Set up the following PNK reaction: Fragmented RNA from step Mix B 3 Mix C 4 Total Gently pipette mix thoroughly and incubate at 37 C for 1 hour and then place the tube on ice. 22. Purify the RNA using a Zymo RNA Clean & Concentrator 5 kit or equivalent RNA purification process. The general protocol for the Zymo RNA purification is described below. 23. Adjust the sample to 50ul using nuclease free water. 24. Add 100 μl of RNA Binding Buffer and pipette mix thoroughly. 25. Add 150 μl of 100% ethanol and pipette mix thoroughly. 26. Transfer the entire volume to the Zymo-Spin IC column and centrifuge at 12,000 xg for 1 minute. Discard the flow through. 27. Add 400 μl of RNA Prep Buffer to the column and centrifuge at 12,000 xg for 1 minute. Discard the flow through. 28. Add 800 μl of RNA Wash Buffer to the column and centrifuge at 12,000 xg for 30 seconds. Discard the flow through. 29. Add 400 μl of RNA Wash Buffer to the column and centrifuge at 12,000 xg for 30 seconds. Discard the flow through. 30. Centrifuge the column at 12,000 xg for 2 minutes to remove and remaining liquid. Discard the flow through. 31. Place the column into a fresh collection tube. 32. Add 10ul of nuclease free water. Incubate at room temperature for 1 minute. 33. Centrifuge at 10,000 xg for 30 seconds. Transfer the flow through to a PCR tube for the next reaction. Enabling Accurate NGS 5

6 3 Adapter Ligation 34. Assemble the following components in a sterile 200 μl PCR tube: RNA from step 33 4 Mix D 2 Total Gently pipette mix thoroughly and incubate at 70 C for 1 minute and then place the tube on ice. 36. Set up the following 3 Adapter Ligation reaction: Denatured 3 adapter and RNA from step 35 6 Mix E 2 Total Gently pipette mix thoroughly and incubate at 28 C for 1 hour. 38. With the tube remaining on the thermal cycler, add 2 μl of Mix F directly into each sample tube. 39. Gently pipette mix thoroughly and continue to incubate at 28 C for an additional 15 minutes and then place the tube on ice. 5 Adapter Ligation 40. Set up the following 5 Adapter Ligation reaction: 3 Adapter Ligated RNA from step Mix H 2 Mix I 2 Total Gently pipette mix thoroughly and incubate at 28 C for 30 minutes and then place the tube on ice. Ligation Product Purification 42. Purify the RNA using a Zymo RNA Clean & Concentrator 5 kit or equivalent RNA purification process. The general protocol for the Zymo RNA purification is described below. 43. Adjust the sample to 50 μl using nuclease free water. 44. Add 100ul of RNA Binding Buffer and pipette mix thoroughly. 45. Add 150ul of 100% ethanol and pipette mix thoroughly. 46. Transfer the entire volume to the Zymo-Spin IC column and centrifuge at 12,000 xg for 1 minute. Discard the flow through. Enabling Accurate NGS 6

7 47. Add 400 μl of RNA Prep Buffer to the column and centrifuge at 12,000 xg for 1 minute. Discard the flow through. 48. Add 800 μl of RNA Wash Buffer to the column and centrifuge at 12,000 xg for 30 seconds. Discard the flow through. 49. Add 400 μl of RNA Wash Buffer to the column and centrifuge at 12,000 xg for 30 seconds. Discard the flow through. 50. Centrifuge the column at 12,000 xg for 2 minutes to remove and remaining liquid. Discard the flow through. 51. Place the column into a fresh collection tube. 52. Add 10ul of nuclease free water. Incubate at room temperature for 1 minute. 53. Centrifuge at 10,000 xg for 30 seconds. Transfer the flow through to a PCR tube for the next reaction. cdna Synthesis 54. Set up the following cdna Synthesis reaction on a new sterile 200 μl PCR tube on ice. Adapter Ligated RNA from step 53 6 Mix I 2 Mix J 2 Total Gently pipette mix thoroughly and incubate at 50 C for 1 hour and then place the tube on ice. PCR Amplification 56. Set up the following PCR reaction. Use a unique barcode primer for each sample. cdna from step Mix K 38 PCR Primer 1 Barcode Primer 1 Total Gently pipette mix thoroughly and amplify the samples in the thermal cycler using the following PCR cycling conditions: 98 C for 30 seconds 15 cycles of: 98 C for 15 seconds 60 C for 15 seconds 72 C for 60 seconds 72 C for 5 minutes Hold at 10 C Enabling Accurate NGS 7

8 Library Purification 58. Purify the PCR product using Ampure XP beads or equivalent PCR purification process. The general protocol for the Ampure XP purification is described below. 59. Add 50 μl of Ampure XP beads to the sample and pipette mix thoroughly. 60. Incubate the sample at room temperature for 5 minutes. 61. Place the tube on the magnetic stand for 2 minutes and remove the supernatant. Be careful not to touch the beads. 62. With the tube remaining on the magnetic stand, gently at 200 μl of 80% ethanol into the tube without disturbing the beads. Wait 30 seconds before discarding the supernatant. Repeat once. 63. Air dry the beads at room temperature for at least 10 minutes until all excess ethanol has evaporated. 64. Take the tube off of the magnetic stand and resuspend the beads using 22.5 μl of elution buffer. Incubate the sample for 2 minutes. 65. Place the tube on the magnetic stand for 2 minutes or until the solution is completely clear. Transfer the supernatant to a new tube. 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 BioAnalyzer trace of library generated from a human kidney tissue Total RNA Sample Enabling Accurate NGS 8