Ion Total RNA-Seq Kit v2 User Guide (Pub. no. 4476286 ... - URGV

2Chapter 2 Prepare Whole Transcriptome LibrariesConstruct the whole transcriptome libraryc. Add 1 μL of the selected Ion Xpress RNA‐Seq Barcode BC primer (choosefrom BC01–BC16) to each PCR tube.d. Proceed to step 2.2. Flick the tube or slowly pipet the solution up and down 5 times to mix well, thencentrifuge briefly to collect the liquid in the bottom of the tube.3. Run the PCRs in a thermal cycler:Stage Temp TimeHold 94°C 2 minCycle (2 cycles) 94°C 30 sec50°C 30 sec68°C 30 secCycle94°C 30 sec• 16 cycles for 1–5 ng poly(A) RNA or 62°C 30 sec10–100 ng rRNA-depleted RNA• 14 cycles for >5 ng poly(A) RNA or>100 ng rRNA-depleted RNA68°C 30 secHold 68°C 5 minPurify theamplified cDNARequired materials from the Magnetic Bead Cleanup Module• Wash Solution Concentrate• Binding Solution Concentrate• Nucleic Acid Binding Beads• Processing Plate• Nuclease‐Free WaterOther materials and equipment• 100% ethanol or 200 proof (absolute) ethanol, ACS‐grade or higher quality• Magnetic stand for 96‐well plates (Life Technologies, Cat. no. AM10027 orAM10050)• 37°C heat block or water bath• (Optional) MicroAmp ® Clear Adhesive Film (Life Technologies, Cat. no. 4306311)Before you begin• If you have not done so already, add 44 mL of 100% ethanol to the Wash SolutionConcentrate and mix well. Mark the label on the bottle to indicate that you addedethanol. Store the solution at room temperature (15°C to 30°C).• If you see a white precipitate in the Binding Solution Concentrate, warm thesolution at 37°C, then shake the solution to dissolve any precipitate before use.• Incubate the Nuclease‐Free Water at 37°C for ≥5 minutes.Note: To reduce the chance of cross‐contamination, we strongly recommend sealingunused wells on the Processing Plate with MicroAmp ® Clear Adhesive Film (LifeTechnologies, Cat. no. 4306311). You can also skip a row between sample rows.30 Ion Total RNA-Seq Kit v2 User Guide