Nov 16, 2020

Public workspaceRT-free Nanopore direct RNA sequencing v1

DOI
dx.doi.org/10.17504/protocols.io.bpiimkce
  • 1UC Santa Cruz
Miten Jain
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DOI: dx.doi.org/10.17504/protocols.io.bpiimkce
Protocol Citation: Miten Jain 2020. RT-free Nanopore direct RNA sequencing v1. protocols.io https://dx.doi.org/10.17504/protocols.io.bpiimkce
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 have used this protocol in the laboratory and it works for us. We have also used a version of it on the International Space Station for sequencing human poly(A) RNA.
Created: November 08, 2020
Last Modified: November 16, 2020
Protocol Integer ID: 44330
Keywords: nanopore, direct RNA sequencing, RT-free, Oxford Nanopore, NASA, UCSC,
Abstract
Reverse Transcription-free (RT-free) poly(A) RNA protocol for Nanopore Direct RNA Sequencing

This protocol was developed as a collaboration between NASA Houston, Oxford Nanopore Technologies, and UC Santa Cruz to support direct RNA Nanopore sequencing on the International Space Station (ISS).

Contributors:

  • NASA JSC Houston: Sarah Stahl, Aaron S. Burton, Kristen K. John, Sarah L. Castro-Wallace

  • Oxford Nanopore Technologies: Daniel Jachimowicz, Daniel R. Garalde

  • University of California Santa Cruz: Mark Akeson, Benedict Paten, Miten Jain
Materials
This protocol uses the reagents as recommended from the SQK-RNA002 kit from Oxford Nanopore Technologies.
Preparing input RNA:

Record the quality, quantity and size of the input RNA.
Guide criteria:
  • Average fragment size: > 500 bases
  • Mass by Qubit RNA HS assay: Amount500 ng
  • Please ensure there are no detergents or surfactants in the buffer
Transfer Amount500 ng of RNA to a Amount0.2 mL PCR tube.
Adjust the volume to Amount8 µL with nuclease-free water and proceed by adding buffer as shown in the table in Step 2 (below).

Heat and cool RNA:

Reagentof [Stock]Addto [Final]
Poly(A)+ RNAvariable8 µl500 ng or 1.1 pmoles
TE + NaCl buffer (90 mM TRIS pH 6.5, 9 mM EDTA, 450 mM NaCl)9x1 µl1x
TOTAL 9.0 µl 
  • Mix the RNA and buffer by pipetting and spin down.
  • Place the tube in a thermal cycler with the following program: Temperature65 °C for Duration00:01:00 , followed by cooling at Temperature0.1 °C / second to Temperature20 °C . Approximate time for this step is Duration00:07:30 .
  • Transfer RNA to a Amount1.5 mL DNA LoBind Eppendorf tube and proceed by adding to the tube the additional reagents shown in step 3 (below).

8m 30s
RT Adapter (RTA) ligation:
Reagentof [Stock]Addto [Final]
RNA from Step 2 500 ng 9.0 µl 500 ng or 1.1 pmoles
RT Adapter (RTA) 1 µl
RNA Calibration Strand (RCS) (Optional) 50 ng/µl 0.5 µl 25 ng
5x Quick Ligation Reaction Buffer (NEB) 5x 3.0 µl 1x
T4 DNA Ligase High Concentration (NEB) 2000 U/µl 1.5 µl 200 U/µl
TOTAL 15.0 µl

Note
  • If not using RCS, substitute with Amount0.5 µL of NSF water.
  • The high concentration T4 DNA ligase (NEB) is essential for the ligation steps to work at optimal efficiency.

  • Mix by pipetting and spin down.
  • Incubate the reaction for Duration00:10:00 at room temperature.

10m
Cleaning up the ligation reaction using Agencourt RNAClean SPRI beads:
Add Amount27 µL (1.8X) of resuspended RNAClean XP beads to the ligation reaction and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for Duration00:05:00 at room temperature.

Note
The tube can be put on a rack at ambient temperature for Duration00:05:00 in the absence of a hula mixer.



5m
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tube on magnet and wash the beads with Amount150 µL of Wash Buffer (WSB) without disturbing the pellet as described below:
  • Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and form a pellet.
  • Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet.

Remove the WSB using a pipette, and discard.
Spin down and place the tube back on the magnet. Pipette off any residual WSB.
Remove the tube from the magnetic rack and resuspend pellet in Amount20 µL Nuclease-free water.

Incubate for Duration00:05:00 at room temperature.
5m
Pellet beads on magnet until the eluate is clear and colorless.
Transfer eluate to a clean Amount1.5 mL DNA LoBind Eppendorf tube and proceed by adding to the tube the additional reagents shown in step 4.
Nanopore sequencing adapter (RMX) ligation:
Reagentof [Stock]Addto [Final]
RNA from Step 3 20 µl
RNA adapter mix (RMX) 6 µl
5x Quick Ligation Reaction Buffer (NEB) 5x 8 µl 1x
Nuclease-free water 3 µl
T4 DNA Ligase High Concentration (NEB) 2000 U/µl 3 µl 150 U/µl
TOTAL 40 µl

Note
The high concentration T4 DNA ligase (NEB) is essential for the ligation steps to work at optimal efficiency.

  • Mix by pipetting and spin down.
  • Incubate the reaction for 10 minutes at room temperature.
Cleaning up the ligation reaction using Agencourt RNAClean SPRI beads:
Add Amount40 µL (1X) of resuspended RNAClean XP beads to the ligation reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for Duration00:05:00 at room temperature.

Note
The tube can be put on a rack at ambient temperature for Duration00:05:00 in the absence of a hula mixer.


5m
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Add Amount150 µL of the Wash Buffer (WSB) to the beads. Close the tube lid, and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.

Repeat Step 4.5 a total of 2 washes.
Remove the tube from the magnetic rack and resuspend pellet in Amount21 µL Elution Buffer. Incubate for Duration00:10:00 at room temperature.

10m
Pellet beads on magnet until the eluate is clear and colorless.
Transfer elutate to a clean Amount1.5 mL Eppendorf DNA LoBind tube.
Use Amount1 µL of library to quantify using Qubit RNA HS assay.


Note
This is your final library.

Dilute library for loading:

Note
This protocol does not cover flowcell preparation. Please follow the current recommended protocol from Oxford Nanopore Technologies for preparing flowcell (priming and loading) and using software for the sequencing run.

Reagentof [Stock]Addto [Final]
RNA library from Step 4 20 µl
Nuclease-free water 80 µl
RRB 2x 100 µl 1x
TOTAL 200 µl

  • Mix the reagents above a clean Amount1.5 mL Eppendorf DNA LoBind tube and spin down.
  • The prepared library is ready to be loaded onto the flowcell.