- Documentation
- A few important comments before you start
- Step 1: Ligate 3' Adapter
- Step 2: Ligate 5' Adapter
- Step 3: Reverse Transcription
- Step 4: Amplify Libraries
- Step 5: Post-PCR Size Selection/Clean-up
- Sequencing Guidelines
- Normalize and Pool
- Setup Run in Basespace
- Loading the Sequencer
Documentation
This protocol is based on Illumina’s TruSeq Small RNA Library Prep kit, but includes a post-PCR bead purification step adapted from TriLink’s CleanTag Small RNA Library Preparation Kit. Courtesy of the University of Maryland's Bale Lab and Institute for Genome Sciences.
A few important comments before you start
- This kit is optimized for 1 μg of total RNA input in 5 μl nuclease free water. 10-50ng of purified small RNA input can also be used, with 50ng recommended.
- We use a Tapestation to assay the input RNA quality. RNA Integrity Numbers (RIN) of 8 and higher are preferred.
- We quantify the input RNA using a Qubit fluorometer.
- Keep all reagents on ice unless otherwise stated. Do NOT store any reagents from the fridge, especially beads, on ice.
- Have all beads at room temperature at least 30 minutes prior to use.
- Aliquot the reagents that arrive in large quantities (such as bead washing buffer) into 2 mL Eppendorf tubes.
- Use only filter pipette tips and clean your area so it is free of RNases.
- Ensure that the AMPureXP beads are mixed well immediately prior to use.
Step 1: Ligate 3' Adapter
The following items are included in the Illumina kit. We recommend kits be stored as received in the original manufacturer’s boxes and not unpacked to organize in a reagent box. When kits go bad, it is much easier to contain the damage if reagents were stored as received.
NAME | LOCATION | ACTION |
---|---|---|
10mM ATP | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
HML | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
RA3 | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
RA5 | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
RNase Inhibitor | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
STP | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
T4 RNA Ligase | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
T4 RNA Ligase 3, Deletion Mutant | -20C Freezer | Thaw on ice, centrifuge briefly, keep on ice |
NAME | LOCATION | ACTION |
---|---|---|
Ampure XP Beads | 4C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Ultrapure water | -20 Freezer | Thaw at RT, keep on ice |
Fresh 80% Ethanol (200 proof EtOH + MilliQ water) | RT Bench | Mix fresh each day. Low% EtOH negatively affects cleanups. |
RNAse Free RT-PCR Grade Water | -20C Freezer | Thaw at RT |
Post-Style Magnetic Stand for 96 well plates | RT Bench |
Step 2: Ligate 5' Adapter
Step 3: Reverse Transcription
Name | Location | Instructions |
---|---|---|
5X First Strand Buffer | -20 freezer | Thaw on ice, centrifuge briefly, keep on ice |
100mM DTT | -20 freezer | Thaw on ice, centrifuge briefly, keep on ice |
Superscript II Reverse Transcriptase | -20 freezer | Keep frozen until use, keep on ice |
Reagent | Volume |
---|---|
5X First Strand Buffer | 2 ul |
12.5 mM dNTP Mix | 0.5 ul |
100 mM DTT | 1 ul |
RNase Inhibitor | 1 ul |
SuperScript II RTase | 1 ul |
Step 4: Amplify Libraries
Choose the preheat lid option and set to 100°C.
98°C for 30 seconds
11 cycles (11-15 may be optimized) of: 98°C for 10 seconds 60°C for 30 seconds 72°C for 15 seconds
72°C for 10 minutes
4°C hold
Amplification products can vary based on RNA input amount, tissue type, and species. This process was optimized using 1 μg of total RNA from mouse and human brain. If the trace does not include clear and distinct bands, increase the number of PCR cycles (up to 15).
Step 5: Post-PCR Size Selection/Clean-up
This step is adapted from the “One-step AMPure XP bead purification” from the TriLink CleanTag small RNA Library Prep kit. Illumina's original protocol uses a gel purification method instead, which can be more precise in size selection but is more time and labor intensive.
Sequencing Guidelines
Normalize and Pool
Setup Run in Basespace
Loading the Sequencer
- The next step is to dilute and denature the prepared libraries. Illumina’s general guidelines for this on the NextSeq can be found here.
- Illumina’s system guide for the NextSeq, which covers the sequening workflow, can be found here.
- The recommended loading concentration is 1.6pM and 5% PhiX spike-in.