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This protocol is current in a preliminary DRAFT phase. We’ll remove this disclaimer once we’re are more confident that it is reasonably well validated in our hands. Until then, proceed at your own risk.


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.

Prepare the following Illumina reagents:

10mM ATP
-20C Freezer
Thaw on ice, centrifuge briefly, keep on ice
-20C Freezer
Thaw on ice, centrifuge briefly, keep on ice
-20C Freezer
Thaw on ice, centrifuge briefly, keep on ice
-20C Freezer
Thaw on ice, centrifuge briefly, keep on ice
RNase Inhibitor
-20C Freezer
Thaw on ice, centrifuge briefly, keep on ice
-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

Prepare the following non-Illumina materials:

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

Start LIGATE3-70 program on thermal cycler to preheat.
In a 200ul PCR tube on ice, combine 1ul RA3 and 1ug total RNA in nuclease free water (5ul).
Pipette to mix, then centrifuge briefly.
Place on thermal cycler and select skip step to start program (70C for 2 minutes, 100C lid).
Place samples on ice.
Prepare the following in a new tube, per sample (overage not included):

3' Adapter Mix

NAMEVolume uL
RNase Inhibitor
T4 RNA Ligase 2, Deletion Mutant
Pipette to mix and centrifuge briefly
Add 4ul to each sample tube, for a total volume of 10ul and pipette to mix.
Place on thermocycler and run LIGATE3-28 (28c for 1 hr).
After the 1hr, remove from thermocycler, add 1ul STP, and pipette to mix.
Place on thermocycler and select skip step to complete the rest of program (28C for 15min).
Remove from thermocycler and place on ice.

Step 2: Ligate 5' Adapter

Preheat thermal cycler to 70C
Add 1.1μl × N samples of RA5 to a new 200 μl PCR tube.
Incubate at 70C for 2 min.
Preheat thermal cycler to 28C
Add 1.1μl × N samples 10mM ATP to the tube of RA5 and pipette to mix.
Add 1.1μl × N samples T4 RNA Ligase to the RA5/ATP mixture and pipette mix.
Add 3 μl each to the RA3/RNA samples from 3’ Ligation Step pipette mix.
Incubate at 28C for 1hr
Remove from the thermal cycler and place on ice.

Step 3: Reverse Transcription

Prepare the following:

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
Preheat thermal cycler to 70C, setting the lid temp to 100C
Make 12.5mM dNTP dilution, per sample (overage already included):

12.5mM dNTP dilution

Stock dNTP mix (25mM)
0.55 ul
Ultrapure water
0.55 ul
Pipette to mix and briefly spin, set aside on ice.
Add 6 μl (of 14 μl total) of each adapter-ligated RNA library to a new 200 μl PCR tube.
Remaining 5' and 3' adapter-ligated RNA can be stored at -80°C for up to 7 days.
Add 1 μl RNA RT Primer (RTP) to the tube of adapter-ligated RNA.
Pipette to mix and brief spin, set aside on ice.
Incubate at 70°C for 2 minutes.
Remove from the thermal cycler and place on ice.
Preheat the thermal cycler to 50°C
Prepare RT Mix, per sample:

RT Mix

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
Pipette to mix and brief spin, set aside on ice.
Add 5.5 μl to each tube of adapter-ligated RNA/primer mix.
Pipette to mix and brief spin, set aside on ice.
Incubate at 50°C for 1 hour.
Place on ice after removal from thermal cycler.

Step 4: Amplify Libraries

Prepare Amp Mix, per sample:

Amp Mix

Ultrapure water
8.5 ul
25 ul
RNA PCR primer (RP1)
2 ul
Pipette to mix and brief spin, set aside on ice.
Add 2 μl of appropriate RNA PCR Primer Index (RPIX) to each reverse transcribed sample.
Add 35.5 μl of PCR master mix to each reverse transcribed sample. Pipette to mix and brief spin, set aside on ice.
Perform PCR as follows:

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

Run each sample on Tapestation with the HSD1000 assay. Below is an example trace.

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).

Safe stopping point. If you are stopping, cap the tube and store at -25°C to -15°C for up to 7 days.

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.

Transfer 50 μL of cDNA libraries to PCR tube strips
Add 90 μL of AMPure XP beads (1.8X) and pipette or vortex thoroughly to mix
Incubate on the bench for 10 min at RT
Place on magnetic stand for 4 min, then remove and discard supernatant w/o disrupting pellet
Wash beads on magnet with 200uL of 80% Ethanol. Wait 30 sec, then remove and discard ethanol.
Repeat wash with an additional 200uL of 80% Ethanol.
Following second wash, allow trace ethanol to dry for 5 min. If bead pellet appears dry or cracked before 5min, remove from magnet and proceed with next steps. Do not over-dry or elution will be inefficient.
Resuspend AMPure XP beads in 20uL of TE buffer (10mM, pH 8) to elute DNA.
Incubate 2 min on bench at RT
Return to magnet for 4 min, then carefully transfer supernatant (17uL) to new tube strips without disturbing beads.

Sequencing Guidelines

Normalize and Pool

Quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library.
Run your samples on Tapestation with either the D1000 or the HSD1000 assay. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
Download our nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. Normalize and pool all your libraries to 4, 2, 1, or 0.5 nM in a LoBind microcentrifuge tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
Quantify your pool on Qubit and enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.

Setup Run in Basespace

Sign into Basespace, then go to the Prep tab, Biological Samples, and select Import Samples on the upper right. Use Illumina’s Sample Import Template to enter information about your samples. The SampleID and Name can be the same, but make sure they are unique for each sample. Species can be left blank. Upload the completed .csv to import your samples.
Continue to Prep Libraries. Select your library prep kit based on the index format used. If you used our plate indexes select “IDT-ILMN Nextera UD Index Set (A or B) for Nextera DNA Flex” If you used another index format, you will need to use a different entry for library prep kit. The your project name as the Plate ID. For each sample, check the box next to it on the left, then drag the sample name to the appropriate index well.
Proceed to Pool Libraries. Select all your samples on the left, then drag and drop in the pool on the right. Name the pool your project name.
Continue to Plan Run. Select NextSeq and name your run your project name. Select Single Read or Paired End Read, then enter the cycle numbers based on your selected kit. For example, if you were doing a run using a High Output 75 Cycle kit, you would select Single Read and enter 72 (for dual 10bp indexes) and 0 for Read 2 Cycles.
Press Sequence to complete planning the run. The run will now be available for selection on the sequencer.

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.