Protocol for TotalScriptâ¢ RNA-Seq Kit

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TotalScript RNA-Seq Kit 3.C. Second Strand Synthesis Briefly centrifuge each tube from Part 3.B prior to use. 1. To each 24 µl reaction, add the following and mix on ice: 1 µl DTT 25 µl 2nd Strand Master Mix 50 µl total reaction volume 2. Incubate the reactions for 1 hour at 16°C. 3. Heat inactivate the reactions for 15 minutes at 80°C. 4. Hold at 4°C. Proceed to TotalScript Library Preparation Procedure (Step 4) or freeze the samples at –20°C. 4. TotalScript Library Prep Kit Procedure Steps 4.A – 4.F use components of the TotalScript Library Prep Kit Component Name TotalScript Tagment Buffer TotalScript Enzyme Gap-Fill Buffer Gap-Fill Enzyme TotalScript PCR Primer Cocktail TotalScript Read 1 Sequencing Primer TotalScript Read 2 Sequencing Primer TotalScript Index Read Sequencing Primer Index 1 TotalScript Control RNA TotalScript Stop Solution Cap Color Green Required Reagents: Agencourt XP Kit (Beckman Coulter, cat. no. A63881) 2X Phusion® High Fidelity PCR Master Mix with HF Buffer (NEB, cat. no. M0531) or 2X Phusion® High Fidelity PCR Master Mix with GC Buffer (NEB, cat. no. M0532) Note: For best results, we recommend non-stick microcentrifuge tubes. 6 www.epicentre.com
TotalScript RNA-Seq Kit 4.A. Tagmentation procedure 1. Briefly centrifuge each tube from 3.C. Then, pipette 39 µl of each reaction into a separate microcentrifuge tube. To each reaction (39 µl), add on ice: 10 µl TotalScript Tagment Buffer 1 µl TotalScript Enzyme 50 µl total reaction volume 2. In a thermocycler with heated lid incubate the samples for: 5 minutes at 55°C. Hold at 4°C. Add 5 μl of TotalScript Stop Solution to each sample. Incubate the samples at room temperature for 5 minutes. The total reaction volume is now 55 µl. 4.B. Purify the reactions using AMPure XP magnetic beads. 1. Prepare at least 500 µl of fresh 80% ethanol solution for each sample. 2. Add 65 μL AMPure beads to each reaction. Mix well and let stand at room temperature for 5 minutes. 3. Place the samples on magnetic stand and capture beads for 5 minutes, then remove unbound material. 4. While still on magnet, wash beads twice with 250 μl of 80% Ethanol. 5. Remove any remaining ethanol and allow the beads to dry for 5 minutes. 6. Remove the tubes from the magnetic stand and resuspend the beads in 15 μl of Elution Buffer (10 mM Tris-HCl; pH 8). 7. Place the tubes on the magnetic stand and capture the beads with magnet for 5 minutes. The tagmented cDNA is now in the Elution Buffer phase. 8. Transfer 14 μl of the eluted, Tagmented DNA to fresh microcentrifuge tubes at room temperature. 4.C. Oligo Replacement Kit components required in Part 4.C Component Name Gap-Fill Buffer Index 1 Gap-Fill Enzyme Cap Color Green 1. Briefly centrifuge each tube prior to use. Then, combine at room temperature: 14 µl purified Tagmented DNA from Step 4.B 4 µl Gap-Fill Buffer 1 µl Index 1* 19 µl total volume * or use an index from the TotalScript Index Kit, Cat. No. TSIDX12910 techhelp@epicentre.com • (800) 284-8474 7
Page 1 and 2: TotalScript RNA-Seq Kit Cat. No. TS
Page 3 and 4: TotalScript RNA-Seq Kit Required Re
Page 5: TotalScript RNA-Seq Kit Important!
Page 9 and 10: TotalScript RNA-Seq Kit 4.F. Purify
Page 11: TotalScript RNA-Seq Kit Appendix C.

Protocol for TotalScriptâ¢ RNA-Seq Kit

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Protocol for TotalScriptâ¢ RNA-Seq Kit