TITLE: RCA-NGS for RNA viruses
AUTHORS: Masayasu Misu, Tomoki Yoshikawa, Satoko Sugimoto, Yuki Takamatsu, Takeshi Kurosu, Yukiteru Ouji, Masahide Yoshikawa, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo

[DESCRIPTION]
This RCA-NGS were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. 

These methods enable whole RNA viral genome sequencing by combining the following techniques:
 1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment
 2) the terminal of viral genome sequence determination by barcoded linkers ligation
 3) Amplification of the viral genomic cDNA using an isothermal DNA amplification technique, such as rolling circle amplification (RCA). 

This method can be exploited to determine any whole RNA viral genomes (i.e., single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes).

[BEFORE_START]
This method was only confirmed to work with the working stocks that contain isolated RNA viruses at least 3.0 × 105  TCID50 per ml.
It is recommended to check no bacterial contamination(e.g., Mycoplasma spp.).

[STEPS]
SECTION: Preparation for virus supernatant
1. Centrifuge the working stock virus to remove debris.
 6000 x g, 10 min 

SECTION: Preparation for virus supernatant
3. Unwanted DNA and RNA mainly originating from the virus-infected cells are digested using  .
SECTION: The viral genomic RNA extraction
4. The viral genomic RNA extraction is performed  using   .
SECTION: Remove unwanted DNA
5. Unwanted DNA mainly from the virus-infected cells in the RNA sample is digested using a   .
SECTION: cSP6-polyA Linker DNA ligation
7. 
The viral RNA is ligated with cSP6-polyA Linker DNA using  .
The RNA is ligated to the 3' end with the barcoded(complementary sequence of SP6 (cSP6)) polyA linker DNA. It is able to identify the 3’ terminal viral genome sequence. The PolyA sequence is required for reverse transcription for ONT kit (SQK-PBK004/ PCS109).
 




SECTION: cSP6-polyA Linker DNA ligation
8. The linker-ligated viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10
  

Fill the sample to 100 μl with 80 μl TE (pH 8.0) and add 100 μl (equal volume) of Buffer RCU.

Eluted the RNA in 10 µL RNase-free H2O and transfer the sample to a 0.2 ml PCR tube.

SECTION: Remove unwanted DNA
6. The viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10.
SECTION: Reverse transcription
9. The viral RNA is reverse transcribed using   .
5′ phosphorylated SP6 primer is used for reverse transcription.
 


SECTION: PCR with barcoding
17. PCR enzyme; 
KOD One PCR Master Mix - TOYOBO Catlog #KMM-101
               or
  


SECTION: PCR with barcoding
18. Add 1 µL   

37 °C 15 min 
80 °C 15 min 
8 °C     ∞
SECTION: PCR with barcoding
18. Purification by   
Prepare AMpure XP beads for use; resuspend by voltexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: PCR with barcoding
18. Qubit amount of DNA   

199 µL 
1 µL  library amplicon

Mix by vortex.

Incubate 2 min Room temperature 

SECTION: The viral genomic RNA extraction
4.1. Add 400 µL of binding buffer (with 4 µL  PolyA carrier RNA).

Mix gently by ~5 times pipetting and flicking thoroughly the tube, and spin down.
Room temperature 10 min 
SECTION: The viral genomic RNA extraction
4.2. Transfer the sample to a High Pure Filter Tube.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral genomic RNA extraction
4.3. Add 500 µL  of  inhibitor removal bo transfer the sample to a High Pure Filter Tube.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral genomic RNA extraction
4.4. Add 450 µL  of  wash buffer.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral genomic RNA extraction
4.5. Add 450 µL  of  wash buffer.
 13000 x g, 1 min and discard the flow-through liquid.

Discard the Collection Tube and insert the Filter Tube into a 1.5 ml tube -  . 

SECTION: The viral genomic RNA extraction
4.6. Add 50 µL  Elution Buffer.
 13000 x g, 1 min

 

SECTION: Remove unwanted DNA
6.1. Add an equal volume 56 µL of Buffer RCU and mix gently.

SECTION: Remove unwanted DNA
6.4. Wash the column by 200 µL Buffer RA3.
 11000 x g, 2 min

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a Nuclease-free Collection Tube(1.5 ml).
SECTION: Remove unwanted DNA
6.2. Transfer the sample to a NucleoSpin RNA XS Column.
 11000 x g, 1 min 
SECTION: Remove unwanted DNA
6.3. Wash the column by 400 µL Buffer RA3.
 11000 x g, 1 min

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a new Collection Tube. 
SECTION: Remove unwanted DNA
6.5. Add 10 µL  RNase-free H2O.
 11000 x g, 1 min
Transfer the sample to a 0.2 ml PCR tube -  .

SECTION: cSP6-polyA Linker DNA ligation
7.1. Total 20 μl reaction

10 µL Purified RNA
1 µL 10 μM the cSP6-polyA linker DNA
2 µL 10X T4 RNA Ligase Reaction Buffer
6 µL 50% PEG8000 solution
1 µL T4 RNA Ligase 2, truncated KQ

Mix gently by pipetting and spin down.
Incubate  25 °C 15 min 

SECTION: Preparation for virus supernatant
2. Transfer 180 µL virus supernatant to a 1.5ml screw cap tube.
SECTION: Preparation for virus supernatant
3.1. Total 201 μl reaction

180 µL virus supernatant
20 µL  10X Micrococcal Nuclease Reaction Buffer
1 µL  Micrococcal nuclease

Mix by pipetting and spin down.
37 °C 60 min 


SECTION: Remove unwanted DNA
5.1. Total 56 μl reaction

50 µL the eluted RNA
5 µL  10X reaction buffer
1 µL  DNase I

Mix gently by pipetting and spin down.
37 °C 30 min 
SECTION: Reverse transcription
9.1. Set up pre-mixture

10 µL  RNA (~ 50ng)
1 µL  50 μM SP6 primer
1 µL  nuclease-free H2O
1 µL  10mM dNTP -   

Mix gently by flicking the tube, and spin down.
65 °C 5 min  and 4 °C 1 min 




SECTION: Reverse transcription
9.2. Total 20 μl reaction

13 µL  pre-mixture sample
4 µL  5X SSIV Buffer
1 µL  100mM DTT
1 µL  RNase OUT -   
1 µL  SuperScript IV Reverse Transcriptase

Mix gently by flicking the tube, and spin down.
 55 °C 10 min 
 80 °C 10 min 
 


17. Purification by   
Prepare AMpure XP beads for use; resuspend by voltexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: PCR with barcoding
17.1. PCR reaction (50 μl/tube) as follows:

5 µL  cDNA
2 µL  LWB (barcoding primer, SQK-PBK004)
18 µL  of nuclease-free water
25 µL  PCR enzyme (KOD One / Q5)

<KOD One PCR Master Mix>
Step                                                 Temperature               Time                       
Heat Activation               98 °C        15 s               

30 cycles of 3 steps
Denaturation                   98 °C        10 s                  
Annealing                        62 °C        5 s     
Extension                        68 °C             5 sec/kb           

                                         8 °C                   ∞ 


<Q5 Hot Start High-Fidelity 2X Master Mix>
Step                                                 Temperature               Time                       
Heat Activation               98 °C        30 s               

30 cycles of 3 steps
Denaturation                   98 °C        10 s              
Annealing                        72 °C        10 s        
Extension                        72 °C           40 sec/kb          

                                        72 °C        2 min      
                                         8 °C                   ∞ 


SECTION: Adaptor Ligation
19. Add 1 µL  of RAP to 11 µL  library

mix gently and incubate 25 °C 5 min 

SECTION: PCR with barcoding
18.1. Add 160 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer
 5 min  Room temperature 

SECTION: PCR with barcoding
18.4. Spin down and pipette off any residual ethanol.
SECTION: PCR with barcoding
18.2. Spin down and pellet on a magnet. wait for 1 min 
pipette off the supernatant
SECTION: PCR with barcoding
18.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: PCR with barcoding
18.3. Wash three times by 500 µL  80 % ethanol and remove the ethanol using a pipette and discard.
SECTION: PCR with barcoding
18.5. Resuspend pellet in 12 µL  Elution Buffer (EB). 
37 °C 3 min  and tapping occasionally.
Incubate on rotor mixer
 7 min
SECTION: PCR with barcoding
18.7. Remove retain 12 µL  elute into a new tube.

SECTION: MinION (PBK004/ PCS109)
20. sequencing for 6 hours
17.1. Add 36 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer
 5 min  Room temperature 
17.2. Spin down and pellet on a magnet. wait for 1 min 
pipette off the supernatant
16.3. Wash twice by 100 µL  80 % ethanol and remove the ethanol using a pipette and discard.
16.4. Spin down and pipette off any residual ethanol.
16.5. Resuspend pellet in 20 µL  TE(pH 8.0). 
37 °C 3 min  and tapping occasionally.
Incubate on rotor mixer
 7 min
16.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
16.7. Remove retain 20 µL  elute into a new tube.
SECTION: (Optional step) Size selection
17. Size selection by   
X0.8 volume of AMPure beads recover more than 200 bp of nucleic acids.
SECTION: (Optional step) Size selection
17.1. Add 16 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer
 5 min  Room temperature 
SECTION: (Optional step) Size selection
16.5. Resuspend pellet in 10 µL  nuclease-free water. 
37 °C 3 min  and tapping occasionally.
Incubate on rotor mixer
 7 min
SECTION: (Optional step) Size selection
17.2. Spin down and pellet on a magnet. wait for 1 min 
pipette off the supernatant
SECTION: (Optional step) Size selection
16.3. Wash twice by 100 µL  80 % ethanol and remove the ethanol using a pipette and discard.
SECTION: (Optional step) Size selection
16.4. Spin down and pipette off any residual ethanol.
SECTION: (Optional step) Size selection
16.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: (Optional step) Size selection
16.7. Remove retain 10 µL  elute into a new tube.
SECTION: RNase H treatment and ethanol precipitation
10.  Add 1 µL    .
37 °C  20 min 

SECTION: RNase H treatment and ethanol precipitation
11. Ethanol precipitation using Dr.GenTLE Precipitation Carrier - Takara Bio Catalog #9094.
SECTION: RNase H treatment and ethanol precipitation
11.1. Add 20 µL  TE(pH8.0) to fill up the sample to 40 µL .
.
SECTION: RNase H treatment and ethanol precipitation
11.2. Add 4 µL  3M CH3COONa (pH5.2) .

SECTION: RNase H treatment and ethanol precipitation
11.3. Add 1 µL  Dr.GenTLE Precipitation Carrier.

SECTION: RNase H treatment and ethanol precipitation
11.4. Add 100 µL 100% ethanol.
SECTION: RNase H treatment and ethanol precipitation
11.5. 13000 x g, 15 min 

SECTION: RNase H treatment and ethanol precipitation
11.6. Discard the supernatant.
SECTION: RNase H treatment and ethanol precipitation
11.7. Wash the pellet with 500 µL 70% ethanol.
13000 x g, 5 min 
SECTION: RNase H treatment and ethanol precipitation
11.9. Dissolve the pellet in 12 µL  nuclease-free H2O.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12. Short cDNA fragment is removed from the viral RNA sample using   
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

 

SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.1. Add 36 µL AMPure XP reagent and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.2. Spin down and pellet on a magnet. 
Wait for 1 min and pipette off the supernatant.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.3. Wash twice by 200 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.4. Spin down and pipette off any residual ethanol.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.5. Resuspend pellet in 20 µL  TE(pH 8.0). 
37 °C 3 min  and tapping occasionally.
Incubate on a rotor mixer.
 7 min
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
12.7. Remove retain 20 µL  elute into a new tube.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13. Size selection of the cDNA sample is performed using   .
X0.8 volume of AMPure beads recovers more than 200 bp of nucleic acids.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.1. Add 16 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.2. Spin down and pellet on a magnet. 
Wait for 1 min and pipette off the supernatant.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.3. Wash twice by 200 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.4. Spin down and pipette off any residual ethanol.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.5. Resuspend pellet in 12 µL  nuclease-free water. 
37 °C 3 min  and tapping occasionally.
Incubate on a rotor mixer.
 7 min
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: (Optional step) Short cDNA fragment removal instead of Ethanol precipitation (#11)
13.7. Remove retain 12 µL  elute into a new tube.
SECTION: Circularization of cDNA
14. The cDNA is circularized using CircLigase II ssDNA Ligase - Biosearch Technologies Catalog #CL9021K.
SECTION: Circularization of cDNA
14.1. Total 20 μl reaction

12 µL  cDNA
2 µL  10X reaction buffer
1 µL  50 mM MnCl2
4 µL 5M Betaine
1 µL CircLigase II
 
Mix by pipetting and spin down.
60 °C 60 min 
80 °C 10 min 

SECTION: Circularization of cDNA
15. Ethanol precipitation using Dr.GenTLE Precipitation Carrier - Takara Catalog #9094.
  
Dissolve the pellet in 10 µL  nuclease-free H2O.
SECTION: RNase H treatment and ethanol precipitation
11.8. Discard the supernatant and dry.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16. cDNA is amplified by Rolling circle amplification (RCA) using  GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24. 

SECTION: T7 endonuclease treatment
19. The amplified cDNA by RCA is digested using   to remove branching.

The following protocol is modified based on the Native barcoding amplicons (with EXP-NBD104, EXPNBD114,and SQK-LSK109) protocol (NBA_9093_v109_revA_12Nov2019) provided by Oxford Nanopore Technologies website. 
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16.1. Total 20 μl reaction

10 µL  cDNA
10 µL 2X denaturation buffer

Mix by pipetting and spin down.
95 °C 3 min 
4 °C 
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16.2. Add 20 μl denatured sample to Ready to go GenomiPhi cake.

30 °C 240 min
65 °C 10 min 

SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17. The cDNA is purified by   
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.1. Add 36 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.2. Spin down and pellet on a magnet and wait for 1 min and pipette off the supernatant.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.3. Wash twice by 200 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.4. Spin down and pipette off any residual ethanol.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.5. Resuspend pellet in 40 µL  nuclease-free H2O. 
37 °C 3 min  and tapping occasionally.
Incubate on a rotor mixer.
 7 min
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17.7. Remove retain 40 µL  elute into a new tube.
SECTION: T7 endonuclease treatment
20. The cDNA is purified using   .
  (Add 54 µL AMPure beads)

Resuspend pellet in 24 µL  nuclease-free H2O. 
SECTION: DNA repair and end-prep
21. The purified cDNA is end-prepped using 
  and
 
SECTION: DNA repair and end-prep
22. The cDNA is purified using   .
  (Add 54 µL AMPure beads)

Resuspend pellet in 30 µL  nuclease-free H2O. 
SECTION: Native barcode ligation
24. The end-prepped cDNA is ligated with native barcode using Native Barcoding Expansion - Oxford Nanopore Technologies Catalog #EXP-NBD104, #EXP-NBD114 and   .
SECTION: Native barcode ligation
25. Add 25 µL TE(pH8.0).
SECTION: Adaptor ligation
28. Pool each barcoded sample into a  0.2ml PCR tube (Total 100–200 fmol).
SECTION: T7 endonuclease treatment
19.1. Total 30 μl reaction

x µL (1.0 μg) DNA
3 µL NEBuffer 2
1.5 µL T7 endonuclease I
25-x µL nuclease-free H2O

Mix by pipetting and spin down.
37 °C 30 min 
SECTION: DNA repair and end-prep
21.1. Total 30 μl reaction

24 µL DNA
1.75 µL NEB Next FFPE DNA repair buffer
1 µL NEB Next FFPE DNA repair Mix
1.75 µL Ultra II end-prep reaction buffer
1.5 µL Ultra II end-prep reaction Mix

Mix by pipetting and spin down.
20 °C 30 min 
65 °C 5 min 
SECTION: Native barcode ligation
24.1. Total 25 μl reaction

x µL DNA(about 400ng)
1.5 µL native barcode
12.5 µL Blunt/TA ligase master mix
11-x µL nuclease-free H2O

Mix by pipetting and spin down.
25 °C 20 min 




SECTION: Native barcode ligation
26. The cDNA is purified using   .

SECTION: Native barcode ligation
26.1. Add 40 µL AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
 5 min  Room temperature 
SECTION: Native barcode ligation
26.2. Spin down and pellet on a magnet. wait for 1 min .
Pipette off the supernatant.
SECTION: Native barcode ligation
26.3. Wash twice by 200 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: Native barcode ligation
26.4. Spin down and pipette off any residual ethanol.
SECTION: Native barcode ligation
26.5. Resuspend pellet in 20 µL  nuclease-free water. 
37 °C 3 min  and tapping occasionally.
Incubate on a rotor mixer.
 7 min
SECTION: Native barcode ligation
26.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: Native barcode ligation
26.7. Remove retain 20 µL  elute into a new tube.
SECTION: Adaptor ligation
29. Adaptor Ligation with pooled samples is performed using
Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-LSK109, Native Barcoding Expansion - Oxford Nanopore Technologies Catalog #EXP-NBD104, #EXP-NBD114 and 
  .

SECTION: Adaptor ligation
29.1. Total 50 μl reaction

x µL DNA (100-200 fmol)
2.5 µL Adaptor Mix II 
10 µL NEB Next Quick Ligation Reaction Buffer(5X)
5 µL Quick T4 DNA ligase
32.5-x µL nuclease-free H2O

mix gently and incubate. 
25 °C 20 min 
SECTION: Adaptor ligation
30. The adaptor-ligated cDNA is purified using   .
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: Adaptor ligation
30.1. Add 80 µL AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
 5 min  Room temperature 
SECTION: Adaptor ligation
30.2. Spin down and pellet on a magnet. Wait for 1 min and pipette off the supernatant.
SECTION: Adaptor ligation
30.3. Wash twice by 200 µL.  Short Fragment Buffer(SFB) and remove the ethanol using a pipette and discard.
SECTION: Adaptor ligation
30.4. Spin down and pipette off any residual SFB.
SECTION: Adaptor ligation
30.5. Resuspend pellet in 12 µL  Elution Buffer (EB) 
37 °C 3 min  and tapping occasionally.
Incubate on a rotor mixer.
 7 min
SECTION: Adaptor ligation
30.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: Adaptor ligation
30.7. Remove retain 12 µL  elute into a new tube.
SECTION: DNA repair and end-prep
23. DNA concentration is measured using a Qubit 4 Fluorometer with   .

199 µL 1X working solution
1 µL  DNA

Mix by vortexing.

Incubate 2 min Room temperature and measure.

 

SECTION: Amplification of cDNA by rolling circle amplification (RCA)
18. DNA concentration is measured using a Qubit 4 Fluorometer with   .

199 µL 1X working solution
1 µL  DNA

Mix by vortexing.

Incubate 2 min Room temperature and measure.

 

SECTION: Native barcode ligation
27. DNA concentration is measured using a Qubit 4 Fluorometer with   .

199 µL 1X working solution
1 µL  DNA

Mix by vortexing.

Incubate 2 min Room temperature and measure.
Convert nanogram(ng) into femtomole(fmol) by a calculator. 
 

SECTION: Adaptor ligation
31. DNA concentration is measured using a Qubit 4 Fluorometer with   .

199 µL 1X working solution
1 µL  DNA

Mix by vortexing.

Incubate 2 min Room temperature and measure.

SECTION: Sequencing by MinION
32. Sequencing according to the manufacturer's instructions.