selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing
Jason D Limberis, Joel Ernst, John Metcalfe, Alina Nalyvayko
Abstract
Non-polyadenylated RNA includes a large subset of crucial regulators of RNA expression and constitutes a substantial portion of the transcriptome, playing essential roles in gene regulation. For example, enhancer RNAs are long non-coding RNAs that perform enhancer-like functions, are bi-directionally transcribed, and usually lack polyA tails. This paper presents a novel method, sel Seq, that selectively removes mRNA and pre-mRNA from samples to enable the selective sequencing of crucial regulatory elements, including non-polyadenylated RNAssuch as long non-coding RNA, enhancer RNA, and non-canonical mRNA.
Before start
Prewarm SuperScript III 10X Buffer to Room temperature
Steps
poly-A tailed cDNA synthesis
Mix the following in a 0.2ml tube
| A | B |
|---|---|
| Component | Volume (μl) |
| Total RNA | 1 |
| Oligo dTs | 1.5 |
| 10 mM dNTP mix | 1.5 |
| Nuclease-free H2O | 10 |
poly-A tailed cDNA reaction synthesis components
Denature sample RNA/primer mixture for 0h 5m 0s at 65°C then cool to 4°C for ≥0h 2m 0s
Spin tube briefly and add the following and mix by pipetting
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X SuperScript III Buffer | 2 |
| 25mM MgCl2 | 4 |
| 0.1M DTT | 2 |
| Superscript III Reverse Transcriptase | 2 |
poly-A tailed cDNA reaction synthesis components
Incubate 50°C for 0h 50m 0s followed by 0h 5m 0s at 85°C to deactivate the enzyme, then cool to 4°C and proceed to the next step
Optional: rRNA depletion
Add in the appropriate rRNA depletion oligos for you sample
Incubate 90°C for 0h 2m 0s and ramp down to 85Room temperature at 0.1°C per second then proceed to the next step
poly-A tailed (and ribosomal) RNA depletion
Add 2µL of RNase H
Incubate 37°C for 0h 20m 0s followed by 0h 5m 0s at 65°C to deactivate the enzyme, then cool it to 4°C and proceed to the next step
poly-A tailed (and ribosomal) DNA depletion
Add in the following components and mix gently by pipetting
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X Turbo DNase Buffer | 4 |
| Turbo DNase | 4 |
| Nuclease-free H2O | 10 |
DNase treatment components
Incubate at 37°C for 0h 30m 0s
Bead cleanup
Add 90 μl (1.8X) of resuspended RNAClean XP Beads to the sample
Mix by pipetting 10x
Incubate 0h 15m 0s at 65On ice
Place on the magnet, allow the beads to aggregate, and remove and discard the supernatant
Add 200µL 80% (v/v) ethanol and incubate (still on the magnet) for 0h 0m 30s
Remove the supernatant
Repeat for a total of 2 washes
Air dry for0h 0m 30s, don't allow the beads to become cracked
Remove the tubes from the magnetic rack
Add 50µL H20 (optionally add-in 1µL RNase inhibitor) and resuspend the beads by pipetting ≥10x
Incubate 0h 5m 0s at 65Room temperature
Place on the magnet, aspirate 50µL of the eluant into a new tube
Optional: One-step RT-qPCR quantification
| A | B |
|---|---|
| Component | Volume (μl) |
| Luna Universal Probe One-Step Reaction Mix (2X) | 5 |
| Luna WarmStart RT Enzyme Mix (20X) | 0.5 |
| TaqMan GAPDH Control Reagents (human; 20x) | 0.5 |
| TaqMan 18S rRNA Control Reagents (eukaryotic; 20x) | 0.5 |
| RNA | 2 |
| Nuclease-free H2O | 1.5 |
Luna RT-qPCR one-step quantification
| A | B | C | D | E |
|---|---|---|---|---|
| Step | Temp (C) | Time (s) | Cycles | Ramp Rate (C/s) |
| Reverse transcription | 55 | 600 | 1 | 2.73 |
| Denaturation | 95 | 60 | 45 | 2.73 |
| Denaturation | 95 | 10 | 2.73 | |
| Amplification | 60 | 30 | 2.11 | |
| Capture | 60 | 0 | – |
Cycle parameters for QuantStudio 3
