Protocol for TotalScript™ RNA-Seq Kit

TotalScript RNA-Seq Kit
Important! If using the Mixed Primer option, dilute the Random Hexamer Primer 1:3 with
nuclease free water.
Note: It is recommended to use the Control RNA as a positive control for library prep. Refer to
Appendix B for Control RNA protocol.
1. Heat denature the RNA and anneal the desired cDNA primers. Combine:
x µl Total RNA (1 ng - 5 ng)
2.5 µl TotalScript Optimized Buffer
x µl cDNA primer:
1 µl of Oligo (dT) Primer OR
1 µl of Random Hexamer Primer OR
2 µl of Mixed Primer (1 µl of Oligo (dT) Primer and 1 µl of 1:3 diluted
Random Hexamer Primer
x µl Nuclease-free water
18 µl total volume
2. Place the samples in a thermocycler with heated lid. Heat for 2 minutes at 65°C,
then hold at 4°C.
3.B. 1st Strand cDNA synthesis
Addition of Actinomycin D to the 1st strand cDNA synthesis reaction is optional. Adding
Actinomycin D can improve the strandedness of the TotalScript library by ≈2%.
1. To each 18 µl reaction from Part 3.A add on ice:
2.5 µl DTT
0.5 µl dNTPs
1 µl RiboGuard RNase Inhibitor
1 µl Actinomycin D (250 ng/µl) or Water
1 µl EpiScript Reverse Transcriptase
24 µl total reaction volume
2. In a thermocycler with heated lid, incubate the samples for:
5 minutes at 25°C
25 minutes at 42°C
15 minutes at 70°C
Hold at 4°C
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