TITLE: RT-free Nanopore direct RNA sequencing v1
AUTHORS: Miten Jain

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Reverse Transcription-free (RT-free) poly(A) RNA protocol for Nanopore Direct RNA Sequencing</span></div><div class = "text-block">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). </div><div class = "text-block">Contributors: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">NASA JSC Houston</span><span>: Sarah Stahl, Aaron S. Burton, Kristen K. John, Sarah L. Castro-Wallace</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Oxford Nanopore Technologies</span><span>: Daniel Jachimowicz, Daniel R. Garalde</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">University of California Santa Cruz</span><span>: Mark Akeson, Benedict Paten, Miten Jain</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Oxford Nanopore Technologies</span><span>: Daniel Jachimowicz, Daniel R. Garalde</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">University of California Santa Cruz</span><span>: Mark Akeson, Benedict Paten, Miten Jain</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. Preparing input RNA:Record the quality, quantity and size of the input RNA.Guide criteria: Average fragment size: > 500 basesMass by Qubit RNA HS assay:  Please ensure there are no detergents or surfactants in the bufferMass by Qubit RNA HS assay: Transfer   of RNA to a  PCR tube.Adjust the volume to  with nuclease-free water and proceed by adding buffer as shown in the table in Step 2 (below).
500 ng
500 ng
0.2 mL
8 µl
?. Heat and cool RNA:   ABCD1Reagentof [Stock]Addto [Final]2Poly(A)+ RNAvariable8 µl500 ng or 1.1 pmoles3TE + NaCl buffer 
(90 mM TRIS pH 6.5, 9 mM EDTA, 450 mM NaCl)9x1 µl1x4TOTAL 9.0 µl Place the tube in a thermal cycler with the following program:  for , followed by cooling at  / second to . Approximate time for this step is Transfer RNA to a  DNA LoBind Eppendorf tube and proceed by adding to the tube the additional reagents shown in step 3 (below).
ABCD1Reagentof [Stock]Addto [Final]2Poly(A)+ RNAvariable8 µl500 ng or 1.1 pmoles3TE + NaCl buffer 
(90 mM TRIS pH 6.5, 9 mM EDTA, 450 mM NaCl)9x1 µl1x4TOTAL 9.0 µl
65 °C
0.1 °C
20 °C
1.5 mL
?. RT Adapter (RTA) ligation:   ABCD1Reagentof [Stock]Addto [Final]2RNA from Step 2  500 ng  9.0 µl  500 ng or 1.1 pmoles  3  RT Adapter (RTA)     1 µl  4RNA Calibration Strand (RCS) (Optional)  50 ng/µl    0.5 µl    25 ng  55x Quick Ligation Reaction Buffer (NEB)  5x    3.0 µl    1x  6T4 DNA Ligase High Concentration (NEB)  2000 U/µl    1.5 µl     200 U/µl  7  TOTAL         15.0 µl<b style="mso-bidi-font-weight:
  normal">       Incubate the reaction for  at room temperature.
ABCD1Reagentof [Stock]Addto [Final]2RNA from Step 2  500 ng  9.0 µl  500 ng or 1.1 pmoles  3  RT Adapter (RTA)     1 µl  4RNA Calibration Strand (RCS) (Optional)  50 ng/µl    0.5 µl    25 ng  55x Quick Ligation Reaction Buffer (NEB)  5x    3.0 µl    1x  6T4 DNA Ligase High Concentration (NEB)  2000 U/µl    1.5 µl     200 U/µl  7  TOTAL         15.0 µl<b style="mso-bidi-font-weight:
  normal">
If not using RCS, substitute with    of NSF water.The high concentration T4 DNA ligase (NEB) is essential for the ligation steps to work at optimal efficiency.
?. Cleaning up the ligation reaction using Agencourt RNAClean SPRI beads:
?. Add  (1.8X) of resuspended RNAClean XP beads to the ligation reaction and mix by pipetting.
27 µl
?. Incubate on a Hula mixer (rotator mixer) for  at room temperature.
The tube can be put on a rack at ambient temperature for   in the absence of a hula mixer.
?. 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  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.
150 µl
?. 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  Nuclease-free water.
20 µl
?. Incubate for  at room temperature.
?. Pellet beads on magnet until the eluate is clear and colorless.
?. Transfer eluate to a clean  DNA LoBind Eppendorf tube and proceed by adding to the tube the additional reagents shown in step 4.
1.5 mL
?. Nanopore sequencing adapter (RMX) ligation:  ABCD1Reagentof [Stock]Addto [Final]2RNA from Step 3       20 µl  3  RNA adapter mix (RMX)    6 µl  45x Quick Ligation Reaction Buffer (NEB)   5x    8 µl     1x  5Nuclease-free water       3 µl       6T4 DNA Ligase High Concentration (NEB)  2000 U/µl    3 µl     150 U/µl  7  TOTAL         40 µl<b style="mso-bidi-font-weight:
  normal">       Mix by pipetting and spin down.Incubate the reaction for 10 minutes at room temperature.
ABCD1Reagentof [Stock]Addto [Final]2RNA from Step 3       20 µl  3  RNA adapter mix (RMX)    6 µl  45x Quick Ligation Reaction Buffer (NEB)   5x    8 µl     1x  5Nuclease-free water       3 µl       6T4 DNA Ligase High Concentration (NEB)  2000 U/µl    3 µl     150 U/µl  7  TOTAL         40 µl<b style="mso-bidi-font-weight:
  normal">
The high concentration T4 DNA ligase (NEB) is essential for the ligation steps to work at optimal efficiency.
?. Cleaning up the ligation reaction using Agencourt RNAClean SPRI beads:
?. Add  (1X) of resuspended RNAClean XP beads to the ligation reaction and mix by pipetting.
40 µl
?. Incubate on a Hula mixer (rotator mixer) for  at room temperature.
The tube can be put on a rack at ambient temperature for   in the absence of a hula mixer.
?. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
?. Add  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.
150 µl
?. Repeat Step 4.5  a total of 2 washes.
?. Remove the tube from the magnetic rack and resuspend pellet in  Elution Buffer. Incubate for  at room temperature.
21 µl
?. Pellet beads on magnet until the eluate is clear and colorless.
?. Transfer elutate to a clean  Eppendorf DNA LoBind tube.
1.5 mL
?. Use  of library to quantify using Qubit RNA HS assay.
1 µl
This is your final library.
?. Dilute library for loading:  ABCD1Reagentof [Stock]Addto [Final]2RNA library from Step 4       20 µl  3Nuclease-free water       80 µl       4RRB  2x    100 µl    1x  5TOTAL       200 µl<b style="mso-bidi-font-weight:
  normal">       Mix the reagents above a clean  Eppendorf DNA LoBind tube and spin down.
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.
ABCD1Reagentof [Stock]Addto [Final]2RNA library from Step 4       20 µl  3Nuclease-free water       80 µl       4RRB  2x    100 µl    1x  5TOTAL       200 µl<b style="mso-bidi-font-weight:
  normal">
1.5 mL