TITLE: Nanopore Transcriptomic Sequencing with C. elegans
AUTHORS: Victoria Yarmey

[DESCRIPTION]
This protocol entails sequencing native RNA of young adult C. elegans using NanoPore sequencing (Oxford NanoPore Technologies, ONT). RNA extraction will be conducted using modified versions of Zymo’s “Direct-zol‱ RNA Miniprep” (Zymo R2050) and ONT’s “Worm (Caenorhabditis elegans) RNA” protocols. Library preparation will be conducted according to ONT’s Direct RNA Sequencing Kit (ONT SQK-RNA004) protocols and sequencing will be conducted on a MinION Mk1C device (Figure 1). Subsequent analysis should be conducted via alignment and assembly by the wf-transciptomes workflow in EPI2ME followed by followed by differential expression analysis with DESeq2 and functional enrichment with Goseq and KOBAS 2.0 in R Studio.


 


[BEFORE_START]
Parent C. elegans worm cultures should be reared on Nematode Growth Medium (NGM) plates with OP50 lawns according to standard protocols (see WormBook’s “Maintenance of C. elegans”), roughly 2-4 days before Day 0. All reagents in the Direct-Zol RNA MiniPrep Kit (Zymo R2051) and ONT Direct RNA Sequencing Kit (ONT SQK-RNA004), including the MinION RNA flow cells (FLO-MIN004RA), should be brought to room temperature on Day 3.

[STEPS]
SECTION: Nematode Culture &amp; Synchronization (Day 0)
1. Culture C. elegans on   plates (NGM agar plates) until adulthood and/or they have begun laying eggs onto the E. coli lawn.

SECTION: Nematode Culture &amp; Synchronization (Day 0)
2. Prepare fresh bleaching solution in a sterile 1.5mL microcentrifuge tube:

  
  
       Volume (μl)     DI Water   250     Bleach   250     1M NaOH   500   
  
SECTION: Nematode Culture &amp; Synchronization (Day 0)
3. Using a P1000, add 1mL of M9+TX  + 0.1% Triton-X-100  (22mM KH2PO4, 42mM NA2HPO4, 89mM NaCl, 1mM MgSO4, 0.1% Triton X-100) to a plate of worms. With the same tip, pipette the solution across the plate multiple times to wash the worms off of the lawn into the solution.

SECTION: Nematode Culture &amp; Synchronization (Day 0)
4. Transfer the worms to a new 1.5mL microcentrifuge tube and centrifuge for 10 sec to pellet the worms.
SECTION: Nematode Culture &amp; Synchronization (Day 0)
5. Remove the supernatant and add the bleaching solution.
SECTION: Nematode Culture &amp; Synchronization (Day 0)
6. Incubate for2 min ( 2-5 minutes), shaking occasionally, until the majority of the worm bodies have been dissolve.


 


SECTION: Nematode Culture &amp; Synchronization (Day 0)
7. Centrifuge for 10-20 sec to pellet. Remove the supernatant and resuspend in M9+TX.
SECTION: Nematode Culture &amp; Synchronization (Day 0)
8. Repeat Step 7 two more times (for a total of 3 washes).
SECTION: Nematode Culture &amp; Synchronization (Day 0)
9. Centrifuge for 10-20 sec to pellet, then remove 990μl of supernatant, leaving 50-100μl in the tube.
SECTION: Nematode Culture &amp; Synchronization (Day 0)
10. Using a P100, resuspend the pellet in the 50-100μl of M9+TX, then transfer to a fresh NGM plate by pipetting droplets of eggs outside of the lawn.
SECTION: Nematode Culture &amp; Synchronization (Day 0)
11. Wait 5 min (5-10 minutes) for the plate to dry.

SECTION: Nematode Culture &amp; Synchronization (Day 0)
12. Store the plate upside-down in a 20°C incubator until the eggs have hatched and reach young adult (roughly 60hr) or another desired age.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
13. Using a P1000, add 1mL of M9+TX to the plate of worms. With the same tip, pipette the solution across the plate multiple times to wash the worms off of the lawn into the solution.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
14. Transfer the worms to a new 1.5mL microcentrifuge tube.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
15. Allow 2 min for the worms to settle to the bottom of the tube.


 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
16. Remove the supernatant and resuspend in M9+TX.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
17. Repeat Steps 2-3 two more times (for a total of 3 washes).
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
18. Remove the supernatant and add 600μl of  

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
19. In a fume hood or BSC, insert a motorized pestle into the tube and use 1-2 sec pulses for 60 sec to break the worm cuticle. Visually inspect the tube under a microscope to confirm lysis.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
20. Add 700μl of 95% ethanol to the lysed sample and mix by gently pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
21. Transfer the sample to a Zymo-Spin IICR Column  in a Collection Tube and centrifuge for 16000 x g, 1 min, 4 °C .

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
22. Transfer the column to a new collection tube and discard the flow-through.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
23.1. Add 500μl of    to the column, then centrifuge at 16000 x g, 1 min, 4 °C. Discard the flow-through.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
23.2. Combine 5μl DNase I (6 U/μl) with 75μl DNA Digestion Buffer in an RNase-free tube and mix by gentle inversion. Add the 80μl solution directly to the column matrix.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
23.3. Incubate at room temperature for 15 minutes.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
24. Add 400μl of Direct-zol RNA PreWash buffer to the column and centrifuge at 16000 x g, 1 min, 4 °C. Discard the flow-through.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
25. Repeat Step 14.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
26.  Add 700μl of   to the column and centrifuge at 16000 x g, 1 min, 4 °C.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
27. Transfer the column to a new RNAse-free tube.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
27.1. Add 50μl of    directly to the column matrix and incubate for 2-5 min.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
27.2. Centrifuge at 16000 x g, 1 min, 4 °C to elute the RNA.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
27.3.  Keep the sample on ice or frozen at -80°C until use.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 1, RNA Extraction
23. Treat with DNase I to remove DNA
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 2, RNA Quantification via NanoDrop
28. Add DI Water to a Kimwipe and wipe down the NanoDrop Spectrophotometer. 
 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 2, RNA Quantification via NanoDrop
29. Add 2μl of DI Water   to the pedestal, then open the NanoDrop software for “Nucleic Acids” and run the initial setup & calibration.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 2, RNA Quantification via NanoDrop
30. Wipe the pedestal and add 2μl of    then run a Blank measurement.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 2, RNA Quantification via NanoDrop
31. Wipe the pedestal and add 2μl of sample. Record the concentration (in ng/μl), A260/A280, and A260/A280.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
32. Prepare Qubit 1X dsRNA HS working solution by diluting Qubit RNA HS reagent 1:200 in Qubit RNA HS buffer from the   in a clean, Nuclease-Free tube.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
33. In a PCR tube, combine 190μl of working solution with 10μl of standard. Label the top of the tube for Standard “1”.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
34. Repeat Step 2 for Standard “2”.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
35. In a PCR tube, combine 199μl of working solution with 1μl of sample.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
36. Vortex the standards and sample tubes for 5 sec, then briefly centrifuge.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
37. Allow the standards and sample to incubate at room temperature for 2 min.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
38. On the Qubit Fluorometer, select 1x dsRNA High Sensitivity then select “Read Standards” and insert Standard #1 into the sample chamber. Select “Run standards”.

 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
39. Replace with Standard #2 and select “Run standards” again. Then select “Next” to proceed.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
40. Replace with the sample tube and enter the sample volume added to the assay tube (1μl) then select “Run sample”. Record the concentration (in ng/μl).
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 3, RNA Quantification via Qubit
41. Your RNA should have a minimum concentration of 125 ng/μl (1 ug in 8μl), an A260/A280 ≥ 2.0 and an A260/A230 of 2.0-2.3.

 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
43. Power on the Mk1C and insert a MinION RNA flow cell (FLO-MIN004RA).

 

 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
44. Log in to the MinKNOW software then select Mk1C in the connection manager. Navigate to the Start page and select “Flow Cell Check” and select “Start” after the flow cell type and ID has been recognized. 
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
45.1. Prepare 1x NEBNext Quick Ligation Reaction Buffer   and T4 DNA Ligase   according to the manufacturer’s instructions and place on ice.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
45.2. Spin down the RT Adapter (RTA), RNA CS (RCS) (if using), and RNA Ligation Adapter (RLA), pipette mix and place on ice.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
45.3. Thaw the Wash Buffer (WSB) and RNA Elution Buffer (REB) at room temperature and mix by vortexing. Then spin down and place on ice.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
45.4. After the flow cell check has finished: Check the number of active pores (a minimum of 800 are needed) and the flow cell health indicators after the check has been completed to ensure there are no issues.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
42. Thaw MinION RNA flow cell (FLO-MIN004RA) and bring to room temperature
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 4, Setup for Library Preparation &amp; Sequencing
45. Thaw and prepare the Direct RNA sequencing (SQK-RNA004) library reagents  while the flow cell check is running:
  

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
46. Prepare 1ug of RNA sample in 8μl of    in a PCR tube. Mix gently by pipetting then briefly spin down.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
47. In the same PCR tube, combine the reagents in the following order (pipetting gently to mix in between):
 
  
   Reagent   Volume (μl)     RNA   8     NEBNext Quick Ligation Reaction Buffer   3     RNA CS (RCS)   0.5     Murine RNAse Inhibitor   1     RT Adapter (RTA)   1     T4 Ligase   1.5     Total   15   
  
  
 
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
48. Spin down then incubate for 10 min  at room temperature.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
49. In a clean 1.5 mL DNA LoBind Eppendorf tube, combine the following reagents together to make the reverse transcription master mix (RTMM):
  
   Reagent   Volume (μl)     Nuclease-Free Water   13     10 mM dNTPs   2     5x Induro RT Reaction Buffer   8     Total   23   
  
  
 
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
50. Transfer the RTMM to the PCR tube containing your adapter-ligated RNA and mix by pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
51. Add 2μl of Induro Reverse Transcriptase to the reaction and mix by pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
52. Place the tube in a thermal cycler and incubate at 60 °C for 30 min, then 70 °C for 10 min, and bring the sample to 4 °C before proceeding to the next step.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
53. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
54. Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
55. Add 72 µl of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
56. Incubate on a Hula mixer (rotator mixer) for 5 min at room temperature.

 


SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
57. Prepare 200μl of  .

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
58. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colorless.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
59. Keep the tube on magnet until the supernatant is clear and colorless before washing the beads with 150 µl of freshly prepared 70% ethanol, as described below:
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
59.1. Keeping the magnetic rack on the benchtop, rotate the tube by 180°. Wait for the beads to migrate towards the magnet and to form a pellet.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
59.2. Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
60. Carefully remove the 70% ethanol using a pipette and discard.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
61. Spin down and place the tube back on the magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off any residual ethanol.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
62. Remove the tube from the magnetic rack and resuspend the pellet in 23 µl  . Incubate for 5 min at room temperature.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
63. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
64. Remove and retain 23 µl of eluate into a clean 5 ml Eppendorf DNA LoBind tube.


 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
65. In the same 1.5 ml Eppendorf DNA LoBind tube, combine the reagents in the following order:  
  



 


SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
66.  Mix by pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
67. Incubate the reaction for 10 min at room temperature.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
68. Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
69. Add 16 µl of resuspended Agencourt RNAClean XP beads to the reaction and mix by pipetting.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
70. Incubate on a Hula mixer (rotator mixer) for 5 min at room temperature.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
71. Spin down the sample and pellet on a magnet. Keep the tube on the magnet for 5 min, and pipette off the supernatant when clear and colourless.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
72. Add 150 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 the beads to pellet for 5 min and pipette off the supernatant when clear and colourless.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
73. Repeat Step 72.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
74. Spin down the tube and replace onto the magnetic rack until the beads have pelleted to pipette off any remaining Wash Buffer (WSB).
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
75. Remove the tube from the magnetic rack and resuspend the pellet in 13 µl RNA Elution Buffer (REB) by the gently flicking the tube. 

Incubate for 10 min at room temperature.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
77. Remove and retain 13 µl of eluate into a clean 5 ml Eppendorf DNA LoBind tube.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
78. Quantify 1 µl of reverse-transcribed and adapted RNA using the  .

 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
79. The reverse-transcribed and adapted RNA is now ready for loading into the flow cell.


 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Library Preparation
76. Pellet the beads on a magnet for 5 min until the eluate is clear and colourless.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
80. Thaw the Sequencing Buffer (SB), Library Solution (LIS), RNA Flush Tether (RFT), and Flow Cell Flush (FCF) at room temperature.

Mix by vortexing and spin down.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
81. To prepare the flow cell priming mix in a clean 1.5 ml Eppendorf DNA LoBind tube, combine the following reagents.

Mix by vortexing and spin down at room temperature.


 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
82. Open the MinION or GridION device lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
83. Slide the flow cell priming port cover clockwise to open the priming port.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
84. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles according to the following:
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
84.1. Set a P1000 pipette to 200 µl
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
84.2.  Insert the tip into the priming port
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
84.3. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
85. Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.

Wait for 5 min.

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
86. During this time, prepare the library for loading by combining the following in a new 1.5 ml Eppendorf DNA LoBind tube:  
  

 


SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
87. Complete the flow cell priming acccording to the following:
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
87.1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
87.2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
88. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
89. Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. 

Ensure each drop flows into the port before adding the next.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
90. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
91. Place the light shield onto the flow cell, as follows:
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
91.1. Carefully place the leading edge of the light shield against the clip. 

 

SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Priming &amp; Loading the Flow Cell
91.2. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Running the Sequencing Reaction
92. Navigate to the Start page on the Mk1C and click “Start Sequencing”.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Running the Sequencing Reaction
93. Fill in the experiment details, such as name and flow cell position and sample ID.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Running the Sequencing Reaction
94. On the Kit page, select the Direct RNA Sequencing Kit (ONT SQK-RNA004).
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Running the Sequencing Reaction
95. On the Run configuration tab, select “super-accurate” base-calling.
SECTION: RNA Extraction &amp; Sequencing (Day 3) - Part 5, Running the Sequencing Reaction
96. Click “Start” on the Review page to start the sequencing run.


 

SECTION: Sequencing Analysis (Day 3+)
97. After the sequencing reaction has finished, concatenate the fastq files on a Linux or Max device using the command prompt.


 


 

SECTION: Sequencing Analysis (Day 3+)
99. Open EPI2ME (on cloud or desktop) and run the concatenated file on the wf-transcriptomes workflow.
SECTION: Sequencing Analysis (Day 3+)
100. If conducting differential expression analysis, prepare a reference set and run the DESeq2 workflow in R Studio.
SECTION: Sequencing Analysis (Day 3+)
101. If conducting functional enrichment with a prepared reference set, run with GOseq and KOBAS 2.0 in R Studio.
SECTION: Sequencing Analysis (Day 3+)
99.1. Sign in to EPI2ME and Navigate to the "Launch" tab.


 

SECTION: Sequencing Analysis (Day 3+)
98. Download the C. elegans reference genome:
SECTION: Sequencing Analysis (Day 3+)
98.1. Go to NCBI's genome browser and search C. elegans.
SECTION: Sequencing Analysis (Day 3+)
98.2. Select the Assembly with the green check-mark next to it (it will be the most recent RefSeq version).


 

SECTION: Sequencing Analysis (Day 3+)
98.3. Click "Download"
SECTION: Sequencing Analysis (Day 3+)
98.4. When prompted, Select "Genome sequences (FASTA)", "Annotation features (GFF)", "Transcripts (FASTA)" as the file types
SECTION: Sequencing Analysis (Day 3+)
98.5. Name the zip file then click "Download". Make sure to extract the folder after downloading.
SECTION: Sequencing Analysis (Day 3+)
99.2. Select the "Transcriptomes" workflow from the list.
SECTION: Sequencing Analysis (Day 3+)
99.3. Click "Run in Cloud" and "Launch".
SECTION: Sequencing Analysis (Day 3+)
99.4. Select the "Input Options" on the left side...

for "FASTQ" select the folder containing the fastq_pass files from your sequencing reaction

for "Reference genome" select the file for the worm reference genome (.fna or .fasta) from NCBI

for "Reference transcriptome" select the file for the worm reference transcriptome (.fna or .fasta) from NCBI

for "Reference annotation" select the file for the worm genome annotation features (.gff) from NCBI

for "direct RNA" set to "True" or click the checkbox


 

SECTION: Sequencing Analysis (Day 3+)
99.5. Select "Launch workflow".