Sep 12, 2025
Version 1
Long-read single-cell genome, transcriptome and open chromatin profiling links genotype to phenotypes. V.1
- Alexandra Pančíková1,
- Ruben Cools1,
- Marios Eftychiou1,
- Margo Aertgeerts1,
- joris Vande Velde1,
- Heidi Segers2,
- Jan Cools1,
- Luuk Harbers1,
- Jonas Demeulemeester1
- 1VIB Center for Cancer Biology, VIB, Leuven, Belgium;
- 2Paediatric Oncology, Department of Oncology, KU Leuven, Leuven, Belgium
Protocol Citation: Alexandra Pančíková, Ruben Cools, Marios Eftychiou, Margo Aertgeerts, joris Vande Velde, Heidi Segers, Jan Cools, Luuk Harbers, Jonas Demeulemeester 2025. Long-read single-cell genome, transcriptome and open chromatin profiling links genotype to phenotypes.. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3wdwpv25/v1
Manuscript citation:
Preprint available: https://www.biorxiv.org/content/10.1101/2025.09.08.674950v1
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: Working
We use this protocol and it's working
Created: May 12, 2025
Last Modified: September 12, 2025
Protocol Integer ID: 218092
Keywords: Long Read, Single Cell, Whole Genome, Gene Expression , ATAC , SPLONGGET, 10X Genomics, cell multiomic, open chromatin info at single cell level, whole genome, cell genome, 10x genomics barcoding, 10x multiome atac, chromatin profiling link, open chromatin info, epigenomic profiles from the same nuclei, gene expression assay, epigenomic profile, single cell level, deeper understanding of cell type, capture of both rna expression, splongget, rna expression, state gene regulation, epigenome, cell type, published 10x protocol
Abstract
The 10X Multiome ATAC + Gene Expression assay permits capture of both RNA expression and epigenomic profiles from the same nuclei for a deeper understanding of cell type or state gene regulation. Here we present a modified version of the published 10X protocol: SPLONGGET ! SPLONGGET captures whole genome, epigenome and open chromatin info at single cell level. It leverages 10X Genomics barcoding, modified library preparation, and fragment-length independent sequencing to unlock comprehensive single-cell multiomics.
Attachments
Guidelines
Notes before start: Use wide bore tips for all steps! Work on ice (4°C) at all times when extracting nuclei.
Troubleshooting
Nuclei Isolation
Nuclei Isolation is done according to the 10xgenomics protocol GC000365: Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing.
- Resuspend nuclei in a minimum 50 µL of 1X Diluted Nuclei Buffer (20X nuclei buffer, PN2000207, provided by 10x Genomics), volume depends on target concentration. 1X Diluted Nuclei Buffer is prepared following the user guide Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).
- Count nuclei and dilute nuclei stock to be in the 970 - 3230 nuclei per µL range in 5 µL total volume using the dilution table below of the single cell Multiome ATAC + Gene Expression user guide_CG000338 Rev F. => Do note targeting less than 3000 nuclei can result in ATAC / Whole Genome and cDNA PCR failure
Chromium Next GEM Long Read Single Cell Multiome ATAC / Whole Genome + Gene Expression
The first three sections are followed exactly as described in the Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev F):
Section 1: Transposition (p27 to p30) ,
Section 2: GEM Generation & Barcoding (p31 to p37)
Section 3: Post GEM Incubation Cleanup (p38 to p42) are performed exactly as described in the User Guide: Chromium Next GEM Single Cell Multiome ATAC + Gene Expression - CG000338 Rev F
Note: In Section 3 you obtained 46 µl purified Sample to start with the modified Pre-Amplification PCR step
Pre-Amplification PCR
GET SPLONGGET!
Equilibrate Following Reagents to Room Temperature:
- Pre-AMP primers [10X: 2000271]
- SPRIselect Reagent [Beckman Coulter: B23318]
Place on Ice
- LongAmp‱ Hot Start Taq 2X Master Mix [NEB: M0533]
Prepare/Obtain
- Buffer EB [Qiagen: 19086]
- 80% Ethanol in Nuclease Free H20 - 1mL per Sample
- Magnetic Separator for PCR strips
Prepare Pre-Amplification Mix + PCR
- Prepare Pre-Amplification Mix on ice!
| A | B | |
| Reagent | µl / Reaction | |
| Post GEM Cleanup Sample | 46 | |
| LongAmp Hot Start Taq 2X Master Mix | 50 | |
| Pre-Amp Primers | 4 | |
| Total Volume | 100 µl |
- Pipette mix and centrifuge briefly to collect reaction at the bottom, before incubating the plate in a thermocycler at:
| A | B | C | |
| Lid Temperature Time | Reaction Volume | Run Time | |
| 105 °C | 100 µl | 30 min | |
| Step | Temperature | Time | |
| 1. | 65°C | 5 min | |
| 2. | 95°C | 3 min | |
| 3. | 95°C | 30 sec | |
| 4. | 63°C | 6 min Go to step 3 repeat 6X (Total 7 cycles) | |
| 5. | 4°C | Hold (Max 18 hours) |
The table recommends a starting point for cycle number optimization for based on Targeted Nuclei Recovery.
Pre-Amplification (1.6X) SPRI Cleanup
Vortex the SPRIselect reagent until fully resuspended. Add 160 µl (1.6X) SPRIselect reagent
to each sample. Pipette mix thoroughly
Incubate 5 min at room temperature
Centrifuge briefly. Place on the magnet pos High until the solution clears
Remove the supernatant
Add 300 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
Add 200 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
After the last ethanol removal - Centrifuge briefly and Place on the magnet pos Low
Remove any remaining ethanol
Remove the tube strip from the magnet. Immediately add 100 µl Buffer EB
Gently pipette mix (pipette set to 90 µl) without introducing bubbles
Incubate 5 min at room temperature
Centrifuge briefly. Place on the magnet pos High until the solution clears.
Transfer 100 µl Pre-AMP sample to a new tube strip
Safe Stop: Store at 4°C for up to 72 h or at −20°C for long term storage, or proceed to the next
step
=> Store remaining pre-amplifcation product at -20°C
ATAC / Whole Genome Amplification
Equilibrate Following Reagents to Room Temperature:
- Sample Index Plate N, Set A [10X: 300427]
- SPRIselect Reagent [Beckman Coulter: B23318]
Place on Ice
- LongAmp‱ Hot Start Taq 2X Master Mix [NEB: M0533]
- SI-PCR Primer B [10X:2000128]
Prepare/Obtain
- Buffer EB [Qiagen: 19086]
- 80% Ethanol in Nuclease Free H20 - 1mL per Sample
- Magnetic Separator for PCR strips
ATAC / Whole Genome Amplification + Sample index
- Prepare Sample Index PCR Mix on ice!
| A | B | |
| Reagent | ul / Reaction | |
| Pre-amplified sample | 40 | |
| LongAmp Hot Start Taq 2X Master Mix | 50 | |
| SI-PCR Primer B | 7,5 | |
| Total Volume | 97,5 µl |
- Pipette mix and centrifuge briefly to collect reaction at the bottom.
- Add 2,5 µl of an individual Sample Index N, Set A to each sample. [Choose the appropriate sample index sets to ensure no sample indices overlap in a multiplexed sequencing run] => Total Volume of ATAC / Whole Genome PCR reaction = 100 µl
- Pipette mix and centrifuge briefly to collect reaction at the bottom, before incubating the plate in a thermocycler at:
| A | B | C | |
| Lid Temperature | Reaction Volume | Run Time | |
| 105°C | 100 µl | 59 min | |
| Step | Temperature | Time | |
| 1. | 95°C | 1 min 30 sec | |
| 2. | 95°C | 30 sec | |
| 3. | 65°C | 6 min Go to step 2 repeat 7X (Total 8 cycles) | |
| 4. | 65°C | 5 min | |
| 5. | 4°C | Hold (Max 72 hours) |
The optimal number of cycles is a trade-off between generating sufficient final mass for library construction and minimizing PCR amplification artifacts. The number of cDNA cycles should also be reduced if large numbers of nuclei are sampled.
Post ATAC / Whole Genome amplification (1.6X) SPRI Cleanup
Vortex the SPRIselect reagent until fully resuspended. Add 160 µl (1.6X) SPRIselect reagent
to each sample. Pipette mix thoroughly
Incubate 5 min at room temperature
Centrifuge briefly. Place on the magnet pos High until the solution clears
Remove the supernatant
Add 300 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
Add 200 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
After the last ethanol removal - Centrifuge briefly and Place on the magnet pos Low
Remove any remaining ethanol
Remove the tube strip from the magnet. Immediately add 32 µl Buffer EB
Gently pipette mix (pipette set to 90 µl) without introducing bubbles
Incubate 10 min at room temperature
Centrifuge briefly. Place on the magnet pos Low until the solution clears.
Transfer 32 µl ATAC / Whole Genome sample to a new tube strip
Measure sample Concentration on Qubit.
Store at 4°C for up to 72 h or at −20°C for long term storage until Oxford Nanopore Ligation sequencing DNA V14 (SQK-LSK114).
Post ATAC / Whole Genome Amplification QC
- Run 1 µl sample (max 5ng) on the Agilent Bioanalyzer High Sensitivity DNA chip to determine fragment size.
- Alternate QC methods like Agilent TapeSation or LabChip can be used as well.
cDNA Amplication
Equilibrate Following Reagents to Room Temperature:
- cDNA primers [10X: 2000089]
- SPRIselect Reagent [Beckman Coulter: B23318]
Place on Ice
- Amp Mix [10X: 2000047/2000103]
Prepare/Obtain
- Buffer EB [Qiagen: 19086]
- 80% Ethanol in Nuclease Free H20 - 1mL per Sample
- Magnetic Separator for PCR strips
cDNA Amplification
- Prepare cDNA amplification PCR Mix on ice!
| A | B | |
| Reagent | µl / Reaction | |
| Pre-amplified sample | 35 | |
| Amp Mix | 50 | |
| cDNA Primers | 15 | |
| Total Volume | 100 µl |
- Pipette mix and centrifuge briefly to collect reaction at the bottom, before incubating the plate in a thermocycler at:
| A | B | C | |
| Lid Temperature | Reaction Volume | Run Time | |
| 105°C | 100 µl | 59 min | |
| Step | Temperature | Time | |
| 1. | 98°C | 3 min | |
| 2. | 98°C | 15 sec | |
| 3. | 63°C | 20 sec | |
| 4. | 72°C | 1 min Go to step 2 repeat 7X (Total 8 cycles) | |
| 5. | 72°C | 2 min | |
| 6. | 4°C | Hold (Max 72 hours) |
Post cDNA amplification (0.6X) SPRI Cleanup
Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect reagent (0.6X) to
each sample and pipette mix thoroughly
Incubate 5 min at room temperature
Place on the magnet pos High until the solution clears.
Remove the supernatant
Add 200 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
Add 200 µl 80% ethanol to the pellet. Wait 30 sec and discard all ethanol
After the last ethanol removal - Centrifuge briefly and Place on the magnet pos Low
Remove any remaining ethanol
Remove the tube strip from the magnet. Immediately add 32 µl Buffer EB
Gently pipette mix (pipette set to 90 µl) without introducing bubbles
Incubate 5 min at room temperature
Centrifuge briefly. Place on the magnet pos Low until the solution clears.
Transfer 32 µl cDNA sample to a new tube strip
Post cDNA Amplification QC
- Run 1 µl sample (max 5 ng) on the Agilent Bioanalyzer High Sensitivity DNA chip to determine fragment size.
Oxford Nanopore (ONT) Library preparation and Sequencing
- ONT Library Preparation
Both ATAC / Whole Genome and non fragmented cDNA samples are library prepped exactly following Oxford Nanopore Ligation sequencing amplicons V14 (SQK-LSK114) protocol:
- LSK114 Library Input amounts were adjusted to 150 fmol
- Short Fragment Buffer (ONT, SFB) was used during library preparation wash steps to retain all fragment sizes
- ONT sequencing
Each LSK114 library is sequenced separately on minimal 1 Oxford Nanopore PromethION FlowCell [FLO-PRO114M] (or latest PromethION FlowCell version).
- Expected Yield
With a yield of ~80-100M reads per flow cell, we typically achieve a coverage of ~4000 transcripts per cell for a library containing ~3,000 cells