Protocol for TotalScriptâ¢ RNA-Seq Kit

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TotalScript RNA-Seq Kit 1. Kit Contents TotalScript cDNA Kit (Cat. Nos. TSCD1296 / TSCD12924) Component Name Volume 6 Reactions 24 Reactions TotalScript 1st Strand Buffer 15 µl 60 μl TotalScript Optimized Buffer 15 µl 60 μl Random Hexamer Primer 6 µl 24 μl Oligo d(T) Primer 6 µl 24 µl DTT 21 µl 84 μl dNTPs 5 µl 12 μl RiboGuard RNase Inhibitor 6 µl 24 µl EpiScript Reverse Transcriptase 6 µl 24 µl TotalScript 2nd Strand Master Mix 150 µl 600 µl Nuclease-Free Water 1 ml 2 X 1.5 ml Storage: Store this kit box and its contents at –20°C. Optional Reagent for TotalScript cDNA Synthesis: Actinomycin D 250 ng/µl (≈0.2 mM) in DMSO; Actinomycin D can improve the strandedness of the TotalScript library by ≈2% (Biovision, cat. no. 1036-50). Cap Color Clear TotalScript Library Prep Kit (Cat. Nos. TSLP1296 / TSLP12924) Component Name Volume 6 Rxn 24 Rxn TotalScript Tagment Buffer 60 µl 240 µl TotalScript Enzyme 6 µl 24 μl Gap-Fill Buffer 24 µl 96 μl Gap-Fill Enzyme 6 µl 24 μl TotalScript PCR Primer Cocktail 6 µl 24 μl TotalScript Read 1 Sequencing Primer 30 µl 100 µl TotalScript Read 2 Sequencing Primer 30 µl 100 µl TotalScript Index Read Sequencing Primer 30 µl 100 µl Index 1 6 µl 24 µl TotalScript Control RNA 10 µl 10 µl TotalScript Stop Solution 300 µl 1.2 ml Storage: Store this kit box and its contents at –20°C. Cap Color Green 2 www.epicentre.com
TotalScript RNA-Seq Kit Required Reagents for TotalScript Library Prep Kit: Agencourt AMPure 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) 10 mM Tris-HCl, pH 8 Optional Reagent: TotalScript Index Kit, Cat No. TSIDX12910, 11 indexes (Refer to Appendix C for Index Sequences) 2. Before starting DNA-Free RNA Treat the RNA sample with DNase I to remove all traces of DNA. Then, remove the DNase I prior to the TotalScript cDNA synthesis procedure. RNA Quality For best results, use intact, non-fragmented RNA samples with RIN >7 as assayed using a Bioanalyzer. Amount of RNA TotalScript is optimized for 1-5 ng of total RNA input, without any prior ribosomal RNA removal or poly(A) enrichment. It is crucial to carefully quantify total RNA prior to beginning. For optimal results, do not exceed the maximum amount of RNA (5 ng). cDNA Primer and Buffer Combination The choice of 1st strand cDNA primer and 1st strand cDNA Buffer to use in the 1st strand cDNA synthesis procedure (step 3) and the attributes of the resulting TotalScript library are dependent on the RNA sample. a) When using Total RNA sample: Follow the procedure in Part 3.A. For best results use the TotalScript Optimized Buffer. Then, use these guidelines for the choice of cDNA primer that best meets your needs. • Oligo (dT) Primer yields 3′-bias libraries with
Page 1: TotalScript RNA-Seq Kit Cat. No. TS
Page 5 and 6: TotalScript RNA-Seq Kit Important!
Page 7 and 8: TotalScript RNA-Seq Kit 4.A. Tagmen
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