Oct 10, 2024
Mycelium RNA Extraction and Library Prep
- Chidrupi Golla1
- 1North Carolina State University
Protocol Citation: Chidrupi Golla 2024. Mycelium RNA Extraction and Library Prep. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn7ppgk5/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
I drafted this protocol
Created: October 03, 2024
Last Modified: October 10, 2024
Protocol Integer ID: 109139
Abstract
The goal of the Mycelium RNA extraction and sequencing protocol is to sequence mycelium RNA. The protocol begins with a method to extract mycelium from soil samples. Following this process, the mycelium can be stored if needed. The next step is to extract the RNA from the mycelium samples. The RNA collected will be prepared for sequencing using a MinION flow cell. The library preparation and sample loading follows the Ligation sequencing kit for cDNA by Oxford Nanopore Technologies. The RNA is reverse transcribed into cDNA and the cDNA is then sequenced.
The Ligation sequencing kit for cDNA does include third-party items that need to be purchased from outside sources.
Some key considerations for this protocol are to maintain the sterility of the sample and to prevent fungal rot. The fungal samples may have other organic materials like wood chips or plant roots. The extraction procedure should prevent contamination from occurring by filtering out large particles. Fungal rot can be prevented by maintaining a temperature range between 20°C to 30°C.
Attachments
Materials
General Equipment and Consumables
- centrifuge
- timer
- Glass beakers
- 50 mL conical tubes
- centrifuge tubes
- 3mL Eppendorf tube
- Deionized water
- Qubit and Nanodrop fluorometers
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
Obtaining Mycelium Samples: Extraction of soil fungal mycelium protocol materials
Equipment
- sieve
- magnetic stirrer
- 1000 µm pore nylon mesh
- 50 µm pore metal sleeve
- 50 µm pore nylon mesh
Consumables
- 45.5% sucrose solution
RNA extraction protocol
Consumables
- TRIzol Reagent from Invitrogen
- chloroform
- isopropanol
Ligation sequencing V14 - Direct cDNA sequencing kit materials
- User-supplied VN Primer, 2 µM
- User-supplied Strand-Switching Primer, 10 µM
- User-supplied PR2 Primer, 10 µM
- NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (NEB, E7180S, or E7180L). Alternatively, you can use the NEBNext products below:
- NEBNext Ultra II End Repair / dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- 10 mM dNTP solution (e.g. NEB N0447)
- LongAmp Taq 2X Master Mix(e.g. NEB M0287)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
- RNaseOUT, 40 U/μl (Life Technologies, cat # 10777019)
- RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen UltraPure BSA 50 mg/ml, AM2616)
Equipment
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Thermal cycler
- Pre-chilled freezer block at -20° C for 200 µl tubes
- Hula mixer
Protocol materials
Maxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
In 2 steps
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Step 20
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
In 2 steps
Before start
This kit contains reagents from a third-party site and not included in the Ligation sequencing V14 - Direct cDNA sequencing kit by Oxford Nanopore Technologies.
Obtaining Mycelium Samples: Extraction of soil fungal mycelium
Obtaining Mycelium Samples: Extraction of soil fungal mycelium
9m
9m
Sieve collected soil samples through a 2-mm sieve to remove any root fragments
Disperse a 5 g soil sample into 100 mL of deionized water
Stir the mixture at 500 rpm using a magnetic stirrer for 00:05:00
5m
Filter the mixture through a 1000 µm pore nylon mesh
Thorough wash the oversized particles with 100 mL of deionized water
Repeat steps 4-5 with the filtrate
Filter the filtrate twice through a 50 µm pore metal sleeve containing a 50 µm pore nylon mesh
Discard the filtrate and collect the oversized particles in a 50 mL tube by rinsing them using 35 mL of deionized water for storage if needed (store at 4℃)
Centrifuge the samples for 00:03:00 at 31000xg
3m
Discard the supernatant
Disperse the pellet by adding 50 mL of 45.5% sucrose solution and hand-shaking vigorously
Centrifuge the solution at 50 x g for 00:01:00
1m
Filter the supernatant through a three-layered 50 µm pore size nylon mesh
Repeat steps 9-13 four more times
Discard the remaining soil pellet
Thoroughly wash the particles collected on the nylon mesh with deionized water. Make sure to rinse out the sucrose solution
Collect the particles into a 3 mL Eppendorf tube and allow them to dry completely
Store between 20-30 °C if needed.
RNA extraction
RNA extraction
Ground the particles into a fine powder in liquid nitrogen
Add 2 mL of TRIzol ReagentThermo Fisher ScientificCatalog #15596026 and ground the sample further until the slurry is thawed
Split the sample into two 2 mL Eppendorf tubes
Incubate at room temperature for 00:05:00
5m
Add 300 µL of chloroform and sahe the tube vigorously for 15 seconds
Incubate at room temperature for 00:03:00
3m
Centrifuge the samples at 13.000 x g for 00:15:00 at 4 °C
15m
Repeat steps 5-7
Precipitate the RNA using 500 µL of isopropanol at -20 °C for 02:00:00
2h
Collect the precipitated RNA through centrifugation at 13000 x g for 00:15:00 at 4 °C
15m
Discard the supernatant and resuspend the RNA pellets
Quantify the RNA using the Qubit and Nanodrop fluorometers
Library Preparation: Reverse transcription and strand-switching
Library Preparation: Reverse transcription and strand-switching
Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.
| Reagent | 1. Thaw at room Temperature | 2. Briefly spin down | 3. Mix well by pipetting | |
| User-supplied VN Primer diluted to 2 µM | ✓ | ✓ | ✓ | |
| User-supplied Strand-Switching Primer diluted to 10 µM | ✓ | ✓ | ✓ | |
| 10 mM dNTP solution | ✓ | ✓ | ✓ | |
| RNaseOUT | Not frozen | ✓ | ✓ | |
| Maxima H Minus Reverse Transcriptase | Not frozen | ✓ | ✓ | |
| Maxima H Minus 5x RT Buffer | ✓ | ✓ | Mix by vortexing |
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Maxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
Prepare the RNA in nuclease-free water.
Add 1 µg of total RNA to a 0.2 ml PCR tube
Adjust the volume to 7.5 µL with nuclease-free water
Mix the tube by flicking
Spin down in a microfuge
Prepare the following reaction in the 0.2 ml PCR tube containing the prepared RNA input:
| Reagent | Volume | |
| RNA input from previous step | 7.5 µl | |
| VN Primer diluted to 2 µM | 2.5 µl | |
| 10 mM dNTPS | 1 µl | |
| Total Volume | 11 µl |
Mix gently by flicking the tube and then spin down
Incubate at 60 °C for 00:05:00 and then snap cool on a pre-chilled freezer block for 00:01:00 .
6m
In a separate tube, mix the following together:
| 5x RT Buffer | 4 µl | |
| RNaseOUT | 1 µl | |
| Nuclease-Free water | 1 µl | |
| Strand-Switching Primer diluted to 10 uM | 2 µl | |
| Total Volume | 8 µl |
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Mix gently by flicking the tube, and spin down.
Add the 8 µL of strand-switching reagents (prepared in steps 6-7) to the 11 µL of snap-cooled mRNA (from steps 2-5). Mix by flicking the tube and spin down.
Incubate at 42 °C for 00:02:00 in the thermal cycler.
2m
Add 1 µl of Maxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741 . The total volume is now 20 µL .
Mix gently by flicking the tube, and spin down.
Incubate using the following protocol using a thermal cycler:
| Cycle Step | Temperature | Time | No. of cycles | |
| Reverse Transcription and Strand-Switching | 42 ℃ | 90 minutes | 1 | |
| Heat inactivation | 85 ℃ | 5 minutes | 1 | |
| Hold | 4 ℃ | ∞ |
Library Preparation: RNA Degradation and Second strand synthesis
Library Preparation: RNA Degradation and Second strand synthesis
- Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.
| Reagent | 1. Thaw at room Temperature | 2. Briefly spin down | 3. Mix well by pipetting | |
| User-supplied PR2 Primer diluted to 10 µM | ✓ | ✓ | ✓ | |
| RNaes Cocktail Enzyme Mix | Not frozen | ✓ | ✓ | |
| LongAmp Taq 2X Master Mix | ✓ | ✓ | ✓ |
Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. Keep the beads at room temperature.
Add 1 µL RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286) to the reverse transcription reaction.
Incubate the reaction for 00:10:00 at 37 °C in a thermal cycler.
10m
Resuspend the AMPure XP beads (AXP) by vortexing.
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 17 µL of resuspended AMPure XP beads (AXP) to the reaction and mix by flicking the tube.
0 µL Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tubes on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Note
Note: If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 20 µL nuclease-free water.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at room temperature.
10m
Briefly spin down the tube and pellet the beads on the magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
Remove and retain 20 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Prepare the following reaction in a 0.2 ml thin-walled PCR tube:
| Reagent | Volume | |
| 2x LongAmp Taq Master Mix | 25 µl | |
| PR2 Primer diluted to 10 μM | 2 µl | |
| Reverse-transcribed sample from above | 20 µl | |
| Nuclease-free water | 3 µl | |
| Total Volume | 50 µl |
Incubate using the following protocol:
| Cycle Step | Temperature | Time | No. of cycles | |
| Denaturation | 94 ℃ | 1 minute | 1 | |
| Annealing | 50 ℃ | 1 minute | 1 | |
| Extension | 65 ℃ | 15 minutes | 1 | |
| Hold | 4 ℃ | ∞ |
Resuspend the AMPure XP beads (AXP) by vortexing.
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 40 µL of resuspended AMPure XP beads (AXP) to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tubes on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Note
If the pellet was disturbed, wait for the beads to pellet again before removing the ethanol.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 21 µL nuclease-free water.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at room temperature.
10m
0 µL Briefly spin down the tube and pellet the beads on the magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
Remove and retain 21 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify the DNA using a Agilent Bioanalyzer and Qubit Fluorometer
Library Preparation: cDNA repair and end-prep
Library Preparation: cDNA repair and end-prep
Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice.
- Combine the following reagents in a 0.2 ml PCR tube:
| Reagent | Volume | |
| cDNA sample | 25 µl | |
| Nuclease-free water | 30 µl | |
| Ultra II End-prep reaction buffer | 7 µl | |
| Ultra II End-prep enzyme mix | 3 µl | |
| Total Volume | 60 µl |
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Using a thermal cycler, incubate at 20 °C for 00:05:00 and 65 °C for 00:05:00 .
10m
Resuspend the AMPure XP Beads (AXP) by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 60 µL of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 61 µL nuclease-free water. Incubate for 00:02:00 at room temperature.
2m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00
1m
Remove and retain 61 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Using a thermal cycler, incubate at 20 °C for 00:05:00 and 65 °C for 00:05:00 .
10m
Resuspend the AMPure XP Beads (AXP) by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 60 µL of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 61 µL nuclease-free water. Incubate for 00:02:00 at room temperature.
2m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
Remove and retain 61 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Using a thermal cycler, incubate at 20 °C for 00:05:00 and 65 °C for 00:05:00 .
10m
Resuspend the AMPure XP Beads (AXP) by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 60 µL of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet until supernatant is clear and colorless. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 61 µL nuclease-free water. Incubate for 00:02:00 at room temperature.
2m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
Remove and retain 61 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Library Preparation: Adapter ligation and clean-up
Library Preparation: Adapter ligation and clean-up
Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.
Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.
In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
Between each addition, pipette mix 10-20 times
| Reagent | Volume | |
| cDNA sample from the previous step | 60 µl | |
| Ligation Adapter (LA) | 5 µl | |
| Ligation Buffer (LNB) | 25 µl | |
| NEBNext Quick T4 DNA Ligase | 10 µl | |
| Total | 100 µl |
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 00:10:00 at room temperature.
10m
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colorless.
Wash the beads by adding 250 µL of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Note
Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in 15 µL Elution Buffer (EB). Spin down and incubate for00:10:00 at room temperature.
10m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µL of the eluted sample using a Qubit fluorometer
Depending on your DNA library fragment size, prepare your final library in 12 µL of Elution Buffer (EB).
| Fragment library length | Flow cell loading amount | |
| Very short (<1 kb) | 100 fmol | |
| Short (1-10 kb) | 35–50 fmol | |
| Long (>10 kb) | 300 ng |
Loading and priming MinION
Loading and priming MinION
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
- To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.
| Reagent | Volume per flow cell | |
| Flow Cell Flush (FCF) | 1,170 µl | |
| Bovine Serum Albumin (BSA) at 50 mg/ml | 5 µl | |
| Flow Cell Tether (FCT) | 30 µl | |
| Total volume | 1,205 µl |
BSA is not required but improves sequencing
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:
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 five minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
| Reagent | Volume per flow cell | |
| Sequencing Buffer (SB) | 37.5 µl | |
| Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using | 25.5 µl | |
| DNA library | 12 µl | |
| Total | 75 µl |
Complete the flow cell priming:
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load200 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: 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.
Close the device lid and set up a sequencing run on MinKNOW
Protocol references
1. Awad A, Pena R. 2023. An improved method for extraction of soil fungal mycelium. MethodsX 11:102477.
2. Schumann U, Smith NA, Wang M-B. 2013. A fast and efficient method for preparation of high-quality RNA from fungal mycelia. BMC Res Notes 6:71.
3. 2023. Ligation sequencing V14 - Direct cDNA sequencing (SQK-LSK114) (DCS_9187_v114_revI_31Jul2024). Oxf Nanopore Technol. https://nanoporetech.com/document/ligation-sequencing-v14-direct-cdna-sequencing. Retrieved 21 September 2024.