Download Ion Total RNA-Seq Kit v2 User Guide (Pub. no. 4476286 Rev. D)

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Chapter 2 Prepare Whole Transcriptome Libraries
Fragment the whole transcriptome RNA
Spike‐In Control Mix concentrations and ratios to assess the dynamic range, lower limit of detection, and fold‐change response of your platform. The following table provides guidelines for how much Spike‐In Control Mix to add to the input RNA for whole transcriptome library preparation. For detailed information, refer to the ERCC RNA Spike‐In Control Mixes User Guide:
Volume of Spike-In Mix 1 or Mix 2 (dilution)†
Amount of Sample RNA
Total RNA
Poly(A) RNA
1 ng
—
1 μL (1:1000)
5 ng
—
5 μL (1:1000)
10 ng
—
1 μL (1:100)
50 ng
—
5 μL (1:100)
100 ng
2 μL (1:1000)
1 μL (1:10)
500 ng
1 μL (1:100)
5 μL (1:10)
1000 ng
2 μL (1:100)
—
5000 ng
1 μL (1:10)
—
† ERCC RNA Spike-In Mix 1, ExFold Spike-In Mix 1, or ExFold Spike-In Mix 2.
ERCC_Analysis Plugin
The ERCC_Analysis plugin is intended to help with ERCC RNA Spike‐in Controls. It enables you to quickly determine whether or not the ERCC results indicate a problem with library preparation or the PGM run.
For more information about the ERCC_Analysis Plugin, refer to the ERCC_Analysis Plugin User Bulletin (Pub. no. 4479068). Fragment the RNA
using RNase III
Use components from the Ion Total RNA‐Seq Kit v2: • Nuclease‐Free Water
• 10X RNase III Reaction Buffer
• RNase III
1. On ice, assemble a reaction for each RNA sample in a 0.2‐mL PCR tube:
Component (add in the order shown)
1. RNA sample and Nuclease-Free Water:
• Poly(A) RNA: 1–500 ng
Volume for
One Reaction
8–10 µL
• rRNA-depleted total RNA: 10–500 ng
• WT Control RNA: 500 ng
2. 10X RNase III Reaction Buffer
1 µL
3. RNase III
1 µL
Total volume
10–12 µL
IMPORTANT! To reduce fragmentation variability, accurately pipet 1 μL of 10X RNase III Reaction Buffer and 1 μL of RNase III to each sample. Do not make a master mix that contains only 10X RNase III Reaction Buffer and RNase III.
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Ion Total RNA-Seq Kit v2 User Guide