Jan 26, 2024
Version 2
selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing V.2
Peer-reviewed method
- Jason D Limberis1,
- Joel Ernst1,
- John Metcalfe1,
- Alina Nalyvayko1
- 1University of California, San Francisco
External link: https://doi.org/10.1371/journal.pone.0289442
Protocol Citation: Jason D Limberis, Joel Ernst, John Metcalfe, Alina Nalyvayko 2024. selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwpk6l5r/v2Version created by Jason D Limberis
Manuscript citation:
Limberis JD, Nalyvayko A, Ernst JD, Metcalfe JZ (2023) selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing. PLOS ONE 18(11): e0289442. https://doi.org/10.1371/journal.pone.0289442
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 26, 2024
Last Modified: January 26, 2024
Protocol Integer ID: 94220
Funders Acknowledgement:
Program for Breakthrough Biomedical Research (PBBR)
Grant ID: NA
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, selSeq, 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.
Materials
Required
SuperScript® III First-Strand Synthesis SystemThermo ScientificCatalog #18080-051
RNase H - 1,250 unitsNew England BiolabsCatalog #M0297L
TURBO DNase 2 U/uLFisher ScientificCatalog #AM2239
Agencourt RNAClean XP Magnetic BeadsBeckman CoulterCatalog #A63987
EthanolContributed by users
A thermocycler and a qPCR machine
A magnetic rack
Optional
Luna Universal Probe One-Step RT-qPCR Kit - 200 rxnsNew England BiolabsCatalog #E3006S
Eukaryotic 18S rRNA Endogenous Control (FAM™/MGB probe, non-primer limited)Thermo FisherCatalog #4333760F
TaqMan™ GAPDH Control Reagents (human)Thermo FisherCatalog #402869
rRNA depletion oligos
Before start
Prewarm SuperScript III 10X Buffer to Room temperature
poly-A tailed cDNA synthesis
poly-A tailed cDNA synthesis
Mix the following in a 0.2ml tube
| A | B | |
| Component | Volume (μl) | |
| Total RNA (1-4ug total) | 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 00:05:00 at 65 °C then cool to 4 °C for ≥00:02:00
7m
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 00:50:00 followed by 00:05:00 at 85 °C to deactivate the enzyme, then cool to 4 °C and proceed to the next step
55m
Optional: rRNA depletion
Optional: rRNA depletion
Add in the appropriate rRNA depletion oligos for you sample
Incubate 90 °C for 00:02:00 and ramp down to Room temperature at 0.1 °C per second then proceed to the next step
2m
poly-A tailed (and ribosomal) RNA depletion
poly-A tailed (and ribosomal) RNA depletion
Add 2 µL of RNase H
Incubate 37 °C for 00:20:00 followed by 00:05:00 at 65 °C to deactivate the enzyme, then cool it to 4 °C and proceed to the next step
25m
poly-A tailed (and ribosomal) DNA depletion
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 00:30:00
30m
Bead cleanup
Bead cleanup
Add 90 μl (1.8X) of resuspended RNAClean XP Beads to the sample
Mix by pipetting 10x
Incubate 00:15:00 at On ice
15m
Place on the magnet, allow the beads to aggregate, and remove and discard the supernatant
Add 200 µL 80 % (v/v) ethanol and incubate (still on the magnet) for 00:00:30
30s
Remove the supernatant
Repeat for a total of 2 washes
Air dry for00:00:30 , don't allow the beads to become cracked
30s
Remove the tubes from the magnetic rack
Add 50 µL H20 (optionally add-in 1 µL RNase inhibitor) and resuspend the beads by pipetting ≥10x
Incubate 00:05:00 at Room temperature
5m
Place on the magnet, aspirate 50 µL of the eluant into a new tube
Optional: One-step RT-qPCR quantification
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