Nanopore Transcriptomic Sequencing with C. elegans

Victoria Yarmey

Oct 04, 2024

Nanopore Transcriptomic Sequencing with C. elegans

DOI

dx.doi.org/10.17504/protocols.io.8epv5r4d6g1b/v1

Victoria Yarmey¹

¹North Carolina State University

Victoria Yarmey

North Carolina State University

DOI: dx.doi.org/10.17504/protocols.io.8epv5r4d6g1b/v1

Protocol Citation: Victoria Yarmey 2024. Nanopore Transcriptomic Sequencing with C. elegans. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r4d6g1b/v1

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: Other

We have not attempted this protocol in full.

Created: October 04, 2024

Last Modified: October 04, 2024

Protocol Integer ID: 109177

Keywords: C. elegans, nanopore sequencing, transcriptomics, RNA sequencing, Direct RNA sequencing, RNA extraction, oxford nanopore sequencing

Disclaimer

This protocol was created as an assignment for BIT 495/595: Portable Genome Sequencing (Fall 2024) at NC State University.

Abstract

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.

Protocol Workflow

Materials

ReagentDirect-zol RNA Miniprep PlusZymo ResearchCatalog #R2070  
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852  
ReagentOxford Nanopore Direct RNA sequencing (SQK-RNA002)Oxford Nanopore TechnologiesCatalog #SQK-RNA002  
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  ReagentNEBNext Quick Ligation Reaction Buffer (5X) - 2.0 mlNew England BiolabsCatalog #B6058S  
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854  
ReagentRNA MinION Flow CellOxford Nanopore TechnologiesCatalog #FLO-MIN004RA  

Protocol materials

ReagentNematode growth media agaroseCatalog #substance_media_ngmaStep 1
 
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852Materials, Step 32
 
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202SMaterials, Step 45.1
 
ReagentFreshly prepared 70% ethanol in nuclease free waterStep 57
 
ReagentDirect-zol RNA Miniprep PlusZymo ResearchCatalog #R2070Materials
 
ReagentTRI ReagentZymo ResearchCatalog #R2050-1-50Step 18
 
ReagentRNA wash buffer (Optional)Zymo ResearchCatalog #R1003-3-24In 2 steps
 
ReagentDNase/RNase-Free WaterZymo ResearchCatalog #W1001-1In 4 steps
 
ReagentRNA MinION Flow CellOxford Nanopore TechnologiesCatalog #FLO-MIN004RAMaterials
 
ReagentM9 solution for nematode cultureStep 3
 
ReagentMilliQ WaterStep 29
 
ReagentOxford Nanopore Direct RNA sequencing (SQK-RNA002)Oxford Nanopore TechnologiesCatalog #SQK-RNA002Materials, Step 45
 
ReagentNEBNext Quick Ligation Reaction Buffer (5X) - 2.0 mlNew England BiolabsCatalog #B6058SMaterials, Step 45.1
 
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854Materials, Step 78

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.

Nematode Culture & Synchronization (Day 0)

20m

Culture C. elegans on ReagentNematode growth media agaroseContributed by usersCatalog #substance_media_ngma  plates (NGM agar plates) until adulthood and/or they have begun laying eggs onto the E. coli lawn.

Prepare fresh bleaching solution in a sterile 1.5mL microcentrifuge tube:

    Volume (μl) 
 DI Water  250 
 Bleach  250 
 1M NaOH  500

Using a P1000, add 1mL of M9+TX ReagentM9 solution for nematode cultureContributed by users + 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.

Transfer the worms to a new 1.5mL microcentrifuge tube and centrifuge for 10 sec to pellet the worms.

Remove the supernatant and add the bleaching solution.

Incubate forDuration00:02:00   ( 2-5 minutes), shaking occasionally, until the majority of the worm bodies have been dissolve.


Note
IMPORTANT: Do not allow more than 5 minutes of incubation in the bleaching solution. The point of this step is to dissolve the bodies of the adult worms but leave the eggs unharmed, which is time-dependent. On the opposite side, if the bodies have not been sufficiently broken up then they could retain residual bleach, which would additionally harm future progeny.

Centrifuge for 10-20 sec to pellet. Remove the supernatant and resuspend in M9+TX.

Repeat Step 7 two more times (for a total of 3 washes).

Centrifuge for 10-20 sec to pellet, then remove 990μl of supernatant, leaving 50-100μl in the tube.

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.

Wait Duration00:05:00   (5-10 minutes) for the plate to dry.

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.

RNA Extraction & Sequencing (Day 3) - Part 1, RNA Extraction

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.

Transfer the worms to a new 1.5mL microcentrifuge tube.

Allow 2 min for the worms to settle to the bottom of the tube.


Note
NOTE: Large worms (young adult & Day 1 adult) should settle quickly (~60 sec). Smaller worms (L4 and below) will take more than 1 min to pellet and should be discarded with the supernatant.

Remove the supernatant and resuspend in M9+TX.

Repeat Steps 2-3 two more times (for a total of 3 washes).

Remove the supernatant and add 600μl of ReagentTRI ReagentZymo ResearchCatalog #R2050-1-50 

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.

Add 700μl of 95% ethanol to the lysed sample and mix by gently pipetting.

Transfer the sample to a Zymo-Spin IICR Column  in a Collection Tube and centrifuge for Centrifigation16000 x g, 4°C, 00:01:00  .

Transfer the column to a new collection tube and discard the flow-through.

Treat with DNase I to remove DNA

Add 500μl of ReagentRNA wash buffer (Optional)Zymo ResearchCatalog #R1003-3-24   to the column, then centrifuge at Centrifigation16000 x g, 4°C, 00:01:00 . Discard the flow-through.

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.

Incubate at room temperature for 15 minutes.

15m

Add 400μl of Direct-zol RNA PreWash buffer to the column and centrifuge at Centrifigation16000 x g, 4°C, 00:01:00 . Discard the flow-through.

Repeat Step 14.

 Add 700μl of ReagentRNA wash buffer (Optional)Zymo ResearchCatalog #R1003-3-24  to the column and centrifuge at Centrifigation16000 x g, 4°C, 00:01:00 .

Transfer the column to a new RNAse-free tube.

Add 50μl of ReagentDNase/RNase-Free WaterZymo ResearchCatalog #W1001-1   directly to the column matrix and incubate for 2-5 min.

Centrifuge at Centrifigation16000 x g, 4°C, 00:01:00  to elute the RNA.

 Keep the sample on ice or frozen at -80°C until use.

RNA Extraction & Sequencing (Day 3) - Part 2, RNA Quantification via NanoDrop

Add DI Water to a Kimwipe and wipe down the NanoDrop Spectrophotometer. 
Equipment
NanoDrop Spectrophotometer
NAME
2000/2000c Spectrophotometers
TYPE
NanoDrop
BRAND
ND2000CLAPTOP
SKU
https://www.thermofisher.com/order/catalog/product/ND2000CLAPTOP
LINK

Add 2μl of DI Water ReagentMilliQ WaterContributed by users  to the pedestal, then open the NanoDrop software for “Nucleic Acids” and run the initial setup & calibration.

Wipe the pedestal and add 2μl of ReagentDNase/RNase-Free WaterZymo ResearchCatalog #W1001-1   then run a Blank measurement.

Wipe the pedestal and add 2μl of sample. Record the concentration (in ng/μl), A260/A280, and A260/A280.

RNA Extraction & Sequencing (Day 3) - Part 3, RNA Quantification via Qubit

10m

Prepare Qubit 1X dsRNA HS working solution by diluting Qubit RNA HS reagent 1:200 in Qubit RNA HS buffer from the ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852  in a clean, Nuclease-Free tube.

In a PCR tube, combine 190μl of working solution with 10μl of standard. Label the top of the tube for Standard “1”.

Repeat Step 2 for Standard “2”.

In a PCR tube, combine 199μl of working solution with 1μl of sample.

Vortex the standards and sample tubes for 5 sec, then briefly centrifuge.

Allow the standards and sample to incubate at room temperature for 2 min.

On the Qubit Fluorometer, select 1x dsRNA High Sensitivity then select “Read Standards” and insert Standard #1 into the sample chamber. Select “Run standards”.

Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
https://www.thermofisher.com/order/catalog/product/Q33238#/Q33238
LINK

Replace with Standard #2 and select “Run standards” again. Then select “Next” to proceed.

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).

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.

Note
IMPORTANT: If you do not meet these QC criteria then you may need to perform an additional
clean & concentrate procedure prior to library preparation.

RNA Extraction & Sequencing (Day 3) - Part 4, Setup for Library Preparation & Sequencing

10m

Thaw MinION RNA flow cell (FLO-MIN004RA) and bring to room temperature

Power on the Mk1C and insert a MinION RNA flow cell (FLO-MIN004RA).

Equipment
MinION Mk1C
NAME
Sequencer
TYPE
Oxford Nanopore Technology
BRAND
MIN-101C
SKU
https://store.nanoporetech.com/minion-mk1c.html
LINK

Note
IMPORTANT: The Direct RNA Sequencing Kit (SQK-RNA004) is only compatible with RNA flow cells (FLO-MIN004RA).

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. 

Thaw and prepare the Direct RNA sequencing (SQK-RNA004) library reagents  while the flow cell check is running:
ReagentOxford Nanopore Direct RNA sequencing (SQK-RNA002)Oxford Nanopore TechnologiesCatalog #SQK-RNA002  

Prepare 1x NEBNext Quick Ligation Reaction Buffer ReagentNEBNext Quick Ligation Reaction Buffer (5X) - 2.0 mlNew England BiolabsCatalog #B6058S  and T4 DNA Ligase ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  according to the manufacturer’s instructions and place on ice.

Spin down the RT Adapter (RTA), RNA CS (RCS) (if using), and RNA Ligation Adapter (RLA), pipette mix and place on ice.

Thaw the Wash Buffer (WSB) and RNA Elution Buffer (REB) at room temperature and mix by vortexing. Then spin down and place on ice.

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.

RNA Extraction & Sequencing (Day 3) - Part 5, Library Preparation

2h 10m

Prepare 1ug of RNA sample in 8μl of ReagentDNase/RNase-Free WaterZymo ResearchCatalog #W1001-1   in a PCR tube. Mix gently by pipetting then briefly spin down.

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 
  
  
 

Spin down then incubate for Duration00:10:00    at room temperature.

10m

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 
  
  
 

Transfer the RTMM to the PCR tube containing your adapter-ligated RNA and mix by pipetting.

Add 2μl of Induro Reverse Transcriptase to the reaction and mix by pipetting.

Place the tube in a thermal cycler and incubate at Temperature60 °C   for Duration00:30:00  , then Temperature70 °C   for Duration00:10:00  , and bring the sample to Temperature4 °C   before proceeding to the next step.

40m

Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the stock of Agencourt RNAClean XP beads by vortexing.

Add 72 µl of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting.

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

Equipment
Hula mixer
NAME
Mixer
TYPE
Invitrogen
BRAND
15920D
SKU
Any rotator mixer
SPECIFICATIONS

Prepare 200μl of ReagentFreshly prepared 70% ethanol in nuclease free waterContributed by users .

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colorless.

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:

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.

Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.

Carefully remove the 70% ethanol using a pipette and discard.

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.

Remove the tube from the magnetic rack and resuspend the pellet in 23 µl ReagentDNase/RNase-Free WaterZymo ResearchCatalog #W1001-1 . Incubate for Duration00:05:00   at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 23 µl of eluate into a clean 5 ml Eppendorf DNA LoBind tube.


Note
NOTE: At this stage, the RT-RNA sample can be stored at -80°C for later use.

In the same 1.5 ml Eppendorf DNA LoBind tube, combine the reagents in the following order:  
  



ReagentVolume (uL)
RT-RNA Sample23
NEBNEXT Quick Ligation Reaction Buffer8
RNA Ligation Adapter (RLA)6
T4 DNA Ligase3
Total40

 Mix by pipetting.

Incubate the reaction for Duration00:10:00   at room temperature.

10m

Resuspend the stock of Agencourt RNAClean XP beads by vortexing.

Add 16 µl of resuspended Agencourt RNAClean XP beads to the reaction and mix by pipetting.

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

Spin down the sample and pellet on a magnet. Keep the tube on the magnet for Duration00:05:00  , and pipette off the supernatant when clear and colourless.

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 Duration00:05:00   and pipette off the supernatant when clear and colourless.

Repeat Step 72.

Spin down the tube and replace onto the magnetic rack until the beads have pelleted to pipette off any remaining Wash Buffer (WSB).

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 Duration00:10:00   at room temperature.

10m

Pellet the beads on a magnet for Duration00:05:00   until the eluate is clear and colourless.

Remove and retain 13 µl of eluate into a clean 5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of reverse-transcribed and adapted RNA using the ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854 .

Note
IMPORTANT: The final concentration should be ideally > 30 ng.

The reverse-transcribed and adapted RNA is now ready for loading into the flow cell.


Note
 IMPORTANT: The RNA library must be sequenced immediately and cannot be stored for later use.

RNA Extraction & Sequencing (Day 3) - Part 5, Priming & Loading the Flow Cell

10m

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.

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.

  Reagent
    Volume (μL)
  
  RNA Flush Tether (RFT)
    30
  
  Flow Cell Flush (FCF)
    1,170
  
  Total
    1,200

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.

Slide the flow cell priming port cover clockwise to open the priming port.

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:

Set a P1000 pipette to 200 µl

 Insert the tip into the priming port

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

Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.

Wait for Duration00:05:00  .

During this time, prepare the library for loading by combining the following in a new 1.5 ml Eppendorf DNA LoBind tube:  
  

  Reagent
    Volume (μl)
  
  Sequencing Buffer (SB)
    37.5
  
  Library Solution (LS)
    25.5
  
  RNA Library
    12
  
  Total
    75
  

Complete the flow cell priming acccording to the following:

Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Mix the prepared library gently by pipetting up and down just prior to loading.

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.

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.

Place the light shield onto the flow cell, as follows:

Carefully place the leading edge of the light shield against the clip. 

Note
NOTE: Do not force the light shield underneath the clip.

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.

RNA Extraction & Sequencing (Day 3) - Part 5, Running the Sequencing Reaction

Navigate to the Start page on the Mk1C and click “Start Sequencing”.

Fill in the experiment details, such as name and flow cell position and sample ID.

On the Kit page, select the Direct RNA Sequencing Kit (ONT SQK-RNA004).

On the Run configuration tab, select “super-accurate” base-calling.

Click “Start” on the Review page to start the sequencing run.


Note
NOTE: The sequencing run may take 1-3 days to run to completion.

Sequencing Analysis (Day 3+)

20m

After the sequencing reaction has finished, concatenate the fastq files on a Linux or Max device using the command prompt.

Note
NOTE: If using a Windows device, you may need 3rd party software to concatenate files.

Note
NOTE: It is not necessary to concatenate files for EPI2ME workflows

Download the C. elegans reference genome:

10m

Go to NCBI's genome browser and search C. elegans.

Select the Assembly with the green check-mark next to it (it will be the most recent RefSeq version).


Note
IMPORTANT: Make sure the reference genome matches the strain you sequenced (available in the "Modifier" column)

Click "Download"

When prompted, Select "Genome sequences (FASTA)", "Annotation features (GFF)", "Transcripts (FASTA)" as the file types

Name the zip file then click "Download". Make sure to extract the folder after downloading.

Open EPI2ME (on cloud or desktop) and run the concatenated file on the wf-transcriptomes workflow.

10m

Sign in to EPI2ME and Navigate to the "Launch" tab.


Note
NOTE: For large analysis projects like transcriptomes, it is recommended to use the Cloud capabilities, which requires the user to have an EPI2ME account.

Select the "Transcriptomes" workflow from the list.

Click "Run in Cloud" and "Launch".

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

Note
NOTE: Diffeerential expression analysis can be conducted in EPI2ME whithin this workflow by navigating to "Differential Expression Options" on the left and editing the expression parameters.

Select "Launch workflow".


Note
NOTE: EPI2ME transcriptome alignment and assembly may take anywhere from 3-10 hours depending on the sample.

If conducting differential expression analysis, prepare a reference set and run the DESeq2 workflow in R Studio.

If conducting functional enrichment with a prepared reference set, run with GOseq and KOBAS 2.0 in R Studio.

Protocol references

[1] Goller C. DNA Extraction with ZymoBIOMICS MiniPrep Kit 2024.

[2] Worm  (Caenorhabditis elegans) RNA. Oxford Nanopore Technologies 2021. https://nanoporetech.comdocument/extraction-method/c-elegans-rna (accessed September 20, 2024).

[3] Direct-zol RNA Miniprep Manual https://files.zymoresearch.com/protocols/_r2050_r2051_r2052_r2053_direct-zol_rna_miniprep.pdf?_gl=1*gtbj9*_gcl_aw*R0NMLjE3MjY3NjI5OTAuQ2p3S0NBandsNi0zQmhCV0Vpd0FwTjZfa2pxQTlZTGZQVGw2OEhrN1BEVU1FYnk0SmJtYlBDTjFMRFpHOVlqWEMyX1o4TFdHaGhVVFF4b0NMUmdRQXZEX0J3RQ..*_gcl_au*MTMyOTYzNjM1My4xNzI0MzUxODk2

[4] Bernard F, Dargère D, Rechavi O, Dupuy D. Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs. Nat Commun 2023;14:1229. https://doi.org/10.1038/s41467-023-36915-0.

[5] Liang Y, Gong Z, Wang J, Zheng J, Ma Y, Min L, et al. Nanopore-Based Comparative Transcriptome Analysis Reveals the Potential Mechanism of High-Temperature Tolerance in Cotton (Gossypium hirsutum L.). Plants 2021;10:2517. https://doi.org/10.3390/plants10112517.

[6] Nanopore sequencing of RNA and cDNA molecules in Escherichia coli n.d. https://rnajournal.cshlp.org/content/28/3/400 (accessed September 20, 2024).

[7] KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases | Nucleic Acids Research | Oxford Academic n.d. https://academic.oup.com/nar/article/39/suppl_2/W316/2507217 (accessed September 20, 2024).

[8] Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Research 2011;39:W316–22. https:/ doi.org/10.1093/nar/gkr483.

[9] Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology 2010;11:R14. https://doi.org/10.1186/gb-2010-11-2-r14.

[10] Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology 2010;11:R14. https://doi.org/10.1186/gb-2010-11-2-r14.

[11] epi2me-labs/wf-transcriptomes 2024. https://github.com/epi2me-labs/wf-transcriptomes

[12] Direct RNA sequencing (SQK-RNA004) (DRS_9195_v4_revD_20Sep2023). Oxford Nanopore Technologies 2023. https://nanoporetech.com/document/direct-rna-sequencing-sqk-rna004 (accessed September 20, 2024).

[13] DESeq2. Bioconductor n.d. http://bioconductor.org/packages/DESeq2/ (accessed September
20, 2024).


	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


	Reagent	Volume (μl)
	Nuclease-Free Water	13
	10 mM dNTPs	2
	5x Induro RT Reaction Buffer	8
	Total	23

	Reagent	Volume (uL)
	RT-RNA Sample	23
	NEBNEXT Quick Ligation Reaction Buffer	8
	RNA Ligation Adapter (RLA)	6
	T4 DNA Ligase	3
	Total	40


	Reagent	Volume (μL)
	RNA Flush Tether (RFT)	30
	Flow Cell Flush (FCF)	1,170
	Total	1,200


	Reagent	Volume (μl)
	Sequencing Buffer (SB)	37.5
	Library Solution (LS)	25.5
	RNA Library	12
	Total	75

Public workspaceNanopore Transcriptomic Sequencing with C. elegans

Nanopore Transcriptomic Sequencing with C. elegans