NEBNext. RNA First Strand Synthesis Module LIBRARY PREPARATION. Instruction Manual. NEB #E7525S/L 24/96 reactions Version 4.

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1 LIBRARY PREPARATION NEBNext RNA First Strand Synthesis Module Instruction Manual NEB #E7525S/L 24/96 reactions Version /18 be INSPIRED drive DISCOVERY stay GENUINE

2 This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals. ISO 9001 Registered Quality Management ISO Registered Environmental Management ISO Registered Medical Devices This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information about commercial rights, please us at While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications. ILLUMINA is a registered trademark of Illumina, Inc. IGEPAL is a registered trademark of Rhodia Operations. Copyright 2018, New England Biolabs, Inc; all rights reserved.

3 NEBNext RNA First Strand Sythesis Module Table of Contents: Applications....2 Protocol A for Use with Total RNA...3 Protocol B for Use with Previously Fragmented RNA....7 Kit Components...9 Revision History The Module Includes: The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7525S) and 96 reactions (NEB #E7525L). All reagents should be stored at 20 C. Colored bullets represent the color of the cap of the tube containing the reagent. (pink) NEBNext First Strand Synthesis Reaction Buffer (pink) Random Primers (pink) ProtoScript II Reverse Transcriptase (pink) Murine RNase Inhibitor Required Materials Not Included: NEBNext Poly(A) mrna Magnetic Isolation Module (NEB #E7490) If you are using the module for Directional RNA Seq, Actinomycin D (Sigma #A1410 dissolved in dimethylsulfoxide [DMSO] to 5 µg/µl) is required. See page 5 for details. NEBNext RNA First Strand Synthesis Module is Designed for Use with the Following: #E6111, NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module #E7550, NEBNext Ultra II Directional RNA Second Strand Synthesis Module #E7442, NEBNext Ultra End Repair/dA-Tailing Module #E7445, NEBNext Ultra Ligation Module #M0543, NEBNext Q5 Hot Start HiFi PCR Master Mix 1

4 Applications: The NEBNext RNA First Strand Synthesis Module contains enzymes and buffers required to convert a broad range of input amounts of RNA into cdna using random priming. The fast, user-friendly workflow has minimal hands-on time and is compatible with upstream Poly(A) mrna enrichment and rrna depletion methods; it is also compatible with downstream second strand cdna synthesis for both directional and non-directional RNA-seq workflows. Single stranded cdna can be used directly for second strand cdna synthesis. Each module component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together with NEB #E6111 or NEB #E7550, #E7445, #E7442, and #M0543 to construct an indexed transcriptome library that is sequenced on an Illumina sequencing platform. For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@ neb.com for further information. 2

5 Protocols Symbols! This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input. Colored bullets indicate the cap color of the reagent to be added Note: Follow steps in Protocol (A) if starting material is intact total RNA. Perform mrna isolation, fragmentation and priming using the NEBNext Poly (A) mrna Magnetic Isolation Module (NEB #E7490). If starting material is previously purified and fragmented RNA, proceed to (B) on page 7. This protocol is optimized for approximately 200 bp inserts. Protocol A for Use with Total RNA Starting Material: Total RNA (DNA free) (10 ng 1 µg) purified mrna ( ng), or ribosomal depleted total RNA ( ng) RNA Purity: The RNA sample should be free of DNA, salts (e.g., Mg 2+, or guanidinium salts), divalent cation chelating agents (e.g., EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol). 1. Preparation of First Strand Reaction Buffer and Random Primer Mix Prepare the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) as follows in a nuclease-free tube: (pink) NEBNext First Strand Synthesis Reaction Buffer 8 µl (pink) NEBNext Random Primers 2 µl Nuclease-free water 10 µl Total Volume 20 µl 2. mrna Isolation, Fragmentation and Priming Starting with Total RNA 2.1. Dilute the total RNA with nuclease-free water to a final volume of 50 μl in a nuclease-free 0.2 ml PCR tube and keep on ice To wash the Oligo dt Beads, add the following to a 1.5 ml nucleasefree tube. If preparing multiple libraries, beads for up to 10 samples can be added to a single 1.5 ml tube for subsequent washes (use magnet NEB #S1506 for 1.5 ml tubes). The purpose of this step is to bring the beads from the storage buffer into the binding buffer. The 2X Binding Buffer does not have to be diluted for this step. 3

6 COMPONENT VOLUME PER ONE LIBRARY Oligo dt Beads d(t)25 20 µl RNA Binding Buffer (2X) 100 µl Total Volume 120 µl Wash the beads by pipetting up and down six times Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes) Remove and discard all of the supernatant from the tube. Take care not to disturb the beads Remove the tube from the magnetic rack Add 100 μl RNA Binding Buffer (2X) to the beads and wash by pipetting up and down six times. If preparing multiple libraries, add 100 µl RNA Binding Buffer (2X) per sample Place the tubes on the magnet and incubate at room temperature until the solution is clear (~2 minutes) Remove and discard the supernatant from the tube. Take care not to disturb the beads Add 50 μl RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50 μl RNA Binding Buffer (2X) per sample. This first binding step removes most of the non target RNA Add 50 μl beads to each RNA sample from Step 2.1. Mix thoroughly by pipetting up and down six times Place the tube in a thermocycler and close the lid. Heat the sample at 65 C for 5 minutes and cool to 4 C with the heated lid set at 75 C to denature the RNA and facilitate binding of the mrna to the beads Remove the tube from the thermocycler when the temperature reaches 4 C Mix thoroughly by pipetting up and down six times. Place the tube on the bench and incubate at room temperature for 5 minutes to allow the mrna to bind to the beads Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes) Remove and discard all of the supernatant. Take care not to disturb the beads Remove the tube from the magnetic rack Wash the beads by adding 200 μl of Wash Buffer to the tube to remove unbound RNA. Gently pipette the entire volume up and down 6 times to mix thoroughly.

7 2.19. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes) Remove and discard all of the supernatant from the tube. Take care not to disturb the beads Remove the tube from the magnetic rack Repeat steps Add 50 μl of Tris Buffer (provided in NEB #E7490 kit) to each tube. Gently pipette up and down 6 times to mix thoroughly Place the tube on the thermocycler. Close the lid and heat the samples at 80 C for 2 minutes, then cool to 25 C with the heated lid set at 90 C to do the first elution of the mrna from the beads Remove the tube from the thermocycler when the temperature reaches 25 C Add 50 μl of RNA Binding Buffer (2X) to the sample to allow the mrna to re-bind to the beads. Mix thoroughly by gently pipetting up and down six times Incubate the tube at room temperature for 5 minutes Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes) Remove and discard the supernatant from the tube. Take care not to disturb the beads Remove the tube from the magnetic rack Wash the beads by adding 200 μl of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly Spin down the tube briefly to collect the liquid from the wall and lid of the tube. Note: It is important to spin down the tube to prevent carryover of the Wash Buffer in subsequent steps Place the tube on the magnet at room temperature until the solution is clear (~2 minutes) Remove and discard all of the supernatant from the tube. Take care not to disturb the beads that contains the mrna. Note: It is important to remove all of the supernatant to successfully fragment the mrna in the subsequent steps. Spin down the tube. Place the tube on the magnetic rack and with a 10 μl tip, remove all of the wash buffer. (Caution: Do not disturb beads that contain the mrna). Avoid letting the beads dry out before adding elution buffer Remove the tube from the magnetic rack. 5

8 Note: The next step provides a fragmentation incubation time resulting in an RNA insert size of ~ 200 nt To elute the mrna from the beads and fragment, add 15.5 μl of the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 1. Pipette up and down six times to resuspend the beads Incubate the sample in a thermocycler with the heated lid set at 105 C as follows: 15 minutes at 94 C Hold at 4 C* *Immediately transfer the tube to ice for 1 minute as soon as it is cool enough to handle (~65 C) Quickly spin down the tube in a microcentrifuge to collect the liquid from the sides of the tube and place on the magnet right away until the solution is clear (~1-2 minutes) Collect the fragmented mrna by transferring 13.5 μl of the supernatant to a nuclease-free 0.2 ml PCR tube. Note 1: If the supernatant volume recovered is less than 13.5 μl for any reason, bring the volume up to 13.5 μl by adding the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 1 and continue with the protocol. Note 2: Avoid transferring any of the magnetic beads Place the tube on ice and proceed directly to First Strand cdna Synthesis. 3. First Strand cdna Synthesis Note: If you are performing first strand synthesis as part of a directional RNA sequence workflow, it is recommended to add Actinomycin D to the reaction. 3.1.! Dilute Actinomycin D stock solution (5 µg/µl) to 0.1 µg/µl in nuclease free water for immediate use. Note: Dilute solutions of Actinomycin D are very sensitive to light. In solution, Actinomycin D tends to adsorb to plastic and glass. For these reasons, unused dilute solutions should be discarded and not stored for further use. However, frozen aliquots of a concentrated stock solution (5 µg/µl) are expected to be stable for at least a month at 20 C. 6

9 To the fragmented and primed mrna (13.5 µl from Step 2.40) add the following components: 3.2A. Non-Directional Reaction Step Up: (pink) Murine RNase Inhibitor 0.5 µl (pink) ProtoScript II Reverse Transcriptase 1 µl Nuclease free water 5 µl Final volume 20 µl 3.2B Directional Reaction Step Up: (pink) Murine RNase Inhibitor 0.5 µl Actinomycin D (0.1 µg/µl) 5 µl (pink) ProtoScript II Reverse Transcriptase 1 µl Final volume 20 µl 3.3. Incubate the sample in a preheated thermocycler with heated lid set at 80 C as follows: 10 minutes at 25 C 50 minutes at 42 C 15 minutes at 70 C Hold at 4 C 3.4. Immediately, perform second strand synthesis reaction, using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB #E6111), or NEBNext Ultra II Directional RNA Second Strand Synthesis Module (NEB #E7550). Protocol B for Use with Previously Fragmented RNA Starting Material: ng of purified mrna fragmented to 200 nt and cleaned up in 13.5 µl of nuclease free water 1. First Strand cdna Synthesis (Non-Directional Reaction Setup) Mix the following components in a sterile PCR tube: Fragmented mrna 13.5 µl Random Primers 1 µl Total volume 14.5 µl 2. Incubate in a preheated thermocycler for 5 minutes at 65 C with heated lid set to 105 C. Hold at 4 C. 3. Spin tube briefly and place on ice 7

10 4. To the fragmented mrna and Random Primers add: First Strand Synthesis Reaction Buffer 4 µl Murine RNase Inhibitor 0.5 µl Total volume 19 µl 5. Incubate in a preheated thermocycler for 2 minutes at 25 C. 6. Add 1 µl ProtoScript II Reverse Transcriptase to the reaction. 7. Incubate the samples in a preheated thermal cycler (with the heated lid set to 80 C) 10 minutes at 25 C 50 minutes at 42 C 15 minutes at 70 C Hold at 4 C 8. Place the tube on ice. 9. Proceed directly to second strand synthesis using NEBNext mrna Second Strand Synthesis Module (NEB #E6111). 8

11 Kit Components NEB #E7525S Table of Components NEB # PRODUCT VOLUME E7421A NEBNext First Strand Synthesis Reaction Buffer ml E7422A Random Primers ml E7423A ProtoScript II Reverse Transcriptase ml E7424A Murine RNase Inhibitor ml NEB #E7525L Table of Components NEB # PRODUCT VOLUME E7421AA NEBNext First Strand Synthesis Reaction Buffer ml E7422AA Random Primers ml E7423AA ProtoScript II Reverse Transcriptase ml E7424AA Murine RNase Inhibitor ml 9

12 Revision History: REVISION # DESCRIPTION DATE 1.0 N/A 2.0 3/ Create "Kit Component Table of Components" for small and large size kits. Delete individual component information pages. 4.0 Add the list of "Designed for Use with the Following". Update protocols. 4/18 12/18 10

13 DNA CLONING DNA AMPLIFICATION & PCR EPIGENETICS RNA ANALYSIS LIBRARY PREP FOR NEXT GEN SEQUENCING PROTEIN EXPRESSION & ANALYSIS CELLULAR ANALYSIS USA New England Biolabs, Inc. 240 County Road Ipswich, MA Telephone: (978) Toll Free: (USA Orders) Toll Free: (USA Tech) Fax: (978) CANADA New England Biolabs, Ltd. Telephone: (905) Toll Free: Fax: (905) Fax Toll Free: CHINA New England Biolabs (Beijing), Ltd. Telephone: / Fax: FRANCE New England Biolabs France Free Call: Free Fax: GERMANY & AUSTRIA New England Biolabs GmbH Telephone: +49/(0)69/ Free Call: 0800/ (Germany) Free Call: 00800/ (Austria) Fax: +49/(0)69/ Free Fax: 0800/ (Germany) JAPAN New England Biolabs Japan, Inc. Telephone: +81 (0) Fax: +81 (0) SINGAPORE New England Biolabs Pte. Ltd. Telephone: Fax: UNITED KINGDOM New England Biolabs (UK) Ltd. Telephone: (01462) Call Free: Fax: (01462) Fax Free: