Oct 17, 2024
Version 2
Modified protocol for genome-wide mapping of uncapped transcripts (GMUCT) in eukaryotes V.2
- Diep R Ganguly1,
- Garima Bhatia1,
- Susheel Sagar Bhat1,
- Brian D Gregory1
- 1University of Pennsylvania
Protocol Citation: Diep R Ganguly, Garima Bhatia, Susheel Sagar Bhat, Brian D Gregory 2024. Modified protocol for genome-wide mapping of uncapped transcripts (GMUCT) in eukaryotes. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2jjbg3p/v2Version created by Diep R Ganguly
Manuscript citation:
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: April 03, 2023
Last Modified: October 17, 2024
Protocol Integer ID: 103663
Keywords: GMUCT, degradome sequencing, RNA decay
Abstract
This is a modified protocol to perform genome-wide mapping of uncapped and cleaved transcripts (GMUCT; Willmann et al, 2014 Methods, 10.1016/j.ymeth.2013.07.003).
Attachments
Guidelines
Make sure your workspace is nuclease-free and sterile, including through the use of clean pipettes, filter tips, microtubes, and PCR tubes to minimize the chance of sample contamination, especially of RNases.
Keep all samples and enzyme mixes on ice in between steps. Be careful for all bead-based size selection steps to avoid unwanted bead carryover.
Materials
| A | B | C | |
| Item | Manufacterer | Catalog Number | |
| Dynabeads mRNA purification kit | Invitrogen | 61006 | |
| NEBNext Poly(A) mRNA Magenetic Isolation module | NEB | E7490 | |
| Magnetic separation rack for microtubes | e.g. Thermo Scientific | e.g. MR02 | |
| Magnetic separation rack for PCR tubes | e.g. VMR | e.g. 10770-240 | |
| HPLC-purified random hexamer primer that includes the TruSeq RA3 3'-adapter on the 5' end: 5'-ctggagttccttggcacccgagaattccannnnnn-3' | IDT | ssDNA oligo order | |
| TruSeq Small RNA adapters and index primers: RA5, RP1, RPI1-48 | Illumina / IDT | ssDNA oligo order | |
| Betaine 5 M | Thermo Scientific Chemicals | J77507.UCR | |
| T4 RNA Ligase 1 (ssRNA Ligase) | NEB | M0204S | |
| Invitrogen Qubit dsDNA HS Assay Kit | Invitrogen | Q32851 | |
| Phusion® High-Fidelity PCR Master Mix with HF Buffer | NEB | M0531S | |
| Superscript II Reverse Trancriptase | Invitrogen | 18064014 | |
| Deoxynucleotide (dNTP) Solution Set | NEB | N0446S | |
| DEPC-treated water | Invitrogen | AM9906 | |
| RNaseOUT Recombinant Ribonuclease Inhibitor | Invitrogen | 10777019 | |
| Adenosine 5'-Triphosphate (ATP) | NEB | P0756S | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q32851 | |
| AMPure XP Beads | Beckman Coulter | A63880 |
Before start
Purify DNA-free total RNA samples using method of choice, for example, TRIzol-chloroform extraction (dx.doi.org/10.17504/protocols.io.bt8wnrxe).
Poly-A selection
Poly-A selection
For each sample, dilute 5 - 30 μg total RNA in 100 μL of nuclease-free water (or 10 mM Tris-Cl pH 6.5).
Perform poly-A RNA selection using Dynabeads mRNA purification kit as per the manufacturer's instructions (see attachments).
Note, the capacity of these beads are 75 μg total RNA. So, if you are starting with < 32.5 μg total RNA, you may want to scale-down all the reactions by one-half in order to conserve reagents.
Determine concentration and purity of poly-A selected RNA using a Nanodrop.
Aliquot a normalized amount of poly-A selected RNA, for 5' adapter, in 9 μL of DEPC-treated water. Ideally, this would be approximately 600 ng poly-A RNA recovered from 30 μg total RNA.
Ligate 5' adapter
Ligate 5' adapter
For each sample, combine the following in PCR tubes and mix by pipetting 6 times.
| A | B | |
| Reagent | Volume (μL) | |
| 25 μM RNA 5' Adapter (RA5) | 1 | |
| Poly-A selected RNA (600 ng) | 9 |
Incubate at 70 ºC for 2 min, then 4 ºC for at least 2 min.
Prepare the following ligation mix, multiplying the volumes for the number of samples being processed (+50% for pipetting errors). Mix with gentle pipetting then briefly centrifuge.
| A | B | |
| Reagent | Volume (μL) | |
| T4 RNA Ligase buffer | 1.5 | |
| 10 mM ATP | 1 | |
| RNaseOUT | 1 | |
| T4 RNA Ligase 1 | 1 |
5' ligation recipe
Add 4.5 μL of the mix to each sample, mix by pipetting 6 times, then briefly spin down.
Incubate at 28 ºC for 1 h.
Proceed directly to the next section or store at -20 ºC overnight.
Poly-A selection
Poly-A selection
Another poly-A selection is performed to remove un-ligated adapter using the NEBNext Poly(A) mRNA Magnetic Isolation Module. This kit is more cost-effective for smaller amounts of RNA.
In a clean PCR tube, aliquot 20 μL of resuspended NEBNext Magnetic Oligo d(T)25 Beads.
Add 100 μL RNA Binding Buffer and pipette mix 6 times.
Place tubes containing beads on a magnetic rack until solution is clear.
Remove supernatant without disturbing the beads.
Remove tubes from the rack and repeat steps 13 - 15.
Resuspend beads in 50 μL RNA Binding Buffer.
Add 35 μL DEPC-treated water to all RNA samples to bring the final volume to 50 μL.
Combine 50 μL of ligated RNA sample with 50 μL beads and mix by pipetting 6 times.
Incubate at 65 ºC for 5 minutes then hold at 4 ºC (denature RNA and facilitate binding of the poly-A to oligo dT beads).
Remove tubes from thermal cycler when samples reach 4 ºC.
Resuspend beads by pipetting 6 times, then incubate for 5 minutes at room temperature.
Repeat step 22.
Place tubes on magnetic rack until solution clear (poly-A RNA bound to beads).
Discard all supernatant without disturbing beads.
Remove tubes from magnetic rack.
Add 200 μL Wash Buffer and mix by pipetting 6 times.
Place tubes on the rack and wait for the solution to become clear.
Discard the supernatant then remove tubes from the rack.
Repeat steps 27 - 29.
Resuspend beads in 8 μL of Tris Buffer and mix by pipetting 6 times.
Incubate samples at 80 ºC for 2 minutes, then hold at 25 ºC.
Once samples reach 25 ºC, immediately place PCR tubes on the magnetic rack.
Once solution is completely clear, transfer the supernatant to a clean nuclease-free PCR Tube.
Continue with reverse transcription reaction or store at -20 ºC.
Reverse transcription and addition of 3' adapter
Reverse transcription and addition of 3' adapter
Set up the following program in a thermal cycler:
- 65 °C for 5 min
- Hold at 4 °C
- 25 °C for 10 min
- 42 °C for 60 min
- 85 °C for 5 min
- Hold at 4 °C
Run the program to allow the thermal cycler to preheat (pause on step 1).
Prepare the following in clean 200 μL PCR tubes for each sample
| A | B | |
| Reagent | Volume (uL) | |
| 5' adapter-ligated RNA | 8 | |
| 20 uM random hexamer primer that includes TruSeq RA3 on the 5' end of the primer (see Materials) | 1 | |
| 12.5 mM dNTPs | 1 |
Mix contents of each tube by pipetting 6 times.
Place tubes in pre-heated thermal cycler and run steps 1-2, allows samples to sit at 4 °C for at least 2 minutes.
Prepare the following reverse transcription mix. Multiply volumes per sample and add 5% to account for pipetting errors. Mix gently by pipetting, then briefly centrifuge.
| A | B | |
| Reagent | Volume (uL) | |
| DEPC-treated water | 2 | |
| 5x First Strand Buffer | 4 | |
| 100 mM DTT | 2 | |
| SuperScript II | 1 | |
| RNaseOUT | 1 |
Reverse transcription recipe
Add 10 μL of the mix to each sample (keep on ice) and mix by pipetting up and down 6 times, then centrifuge briefly.
Return samples to the thermal cycler and continue with steps 3-6.
Size selection
Size selection
Add 20 μL of AMPure XP beads to reverse transcription reaction (1X ratio).
Mix by pipetting six times and incubate at room temperature for 1 minute.
Place tube on magnetic rack and allow solution to become clear.
Remove supernatant.
Add 180 μL of 80 % (v/v) ethanol and mix by pipetting 6 times.
Place tube on magnetic rack and allow solution to clear.
Remove supernatant.
Resuspend beads in 21 μL of nuclease-free water or Tris buffer and incubate for 1 minute at room temperature (off of the magnetic rack)
Place tube on magnetic rack and allow solution to become clear.
Transfer 20 μL of supernatant to a clean tube to setup PCR amplification.
PCR amplification
PCR amplification
Use the Illumina Index Adapter Pooling Guide (TruSeq Small RNA Library Prep Kit) to select which unique RNA PCR Primer Index will be used for each library.
Set up the following program in a thermal cycler:
- 98 °C for 30 sec
- 98 °C for 10 sec
- 60 °C for 30 sec
- 72 °C for 15 sec
- Back to step 2 (11x = 12 cycles total)
- 72 °C for 10 min
- Hold at 4 °C
Run the program to allow the thermal cycler to preheat (pause on step 1).
N.b. For ideal library construction, PCR cycle number should be adjusted to avoid over- (introduce PCR duplicates and artefacts) and under-cycling (low library yield). We aim for our PCRs to run into the early exponential phase (approximately, 12-16 cycles but this will vary depending on input).
Add 2 μL of a unique RPI (10 µM) to each sample and mix by pipetting 6 times.
Prepare the following PCR mix, multiplying the volumes for the number of samples (+50 % for pipetting errors). Mix by pipetting and briefly centrifuge.
| A | B | |
| Reagent | Volume (uL) | |
| 2X Phusion Master Mix | 50 | |
| RNA PCR Primer 1 (RP1) | 2 | |
| 1 M Betaine | 26 |
PCR mix per library
Add 78 μL of the PCR mix to each sample and mix by pipetting up and down 6 times. Centrifuge briefly and keep samples on ice.
Distribute 100 μL across 4 PCR tubes (i.e. 4 x 25 μL reactions per sample). This is done to increase the PCR efficiency.
Place all samples into the pre-heated thermal cycler and run the program.
Size selection
Size selection
Combine 25 μL aliquots into a single 1.5 mL microtube.
Add 1X volume of AMPure XP beads to sample and mix by pipetting 6 times.
Incubate at room temperature for 1 minute.
Place tube on magnetic rack and allow solution to become clear.
Discard supernatant.
Add 180 μL of 80 % (v/v) ethanol and mix by pipetting 6 times.
Place tube on magnetic rack and allow solution to become clear.
Discard supernatant and repeat steps 69 - 70.
Discard supernatant and allow to air-dry for 1 minute.
Resuspend beads in 13 μL nuclease-free water or Tris buffer.
Transfer 12 μL of supernatant into a clean tube. This is your final GMUCT library.
Quantify library concentration using a Qubit fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen). Alternatively, run libraries on a LabChip GXII or Bioanalyzer to give information on concentration and fragment sizes.
Protocol references
M. R. Willmann, N. D. Berkowitz, B. D. Gregory, Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes—GMUCT 2.0. Methods. 67, 64–73 (2014).