Tn5 loading scalebarcodes

Koen Theunis

Jan 12, 2026

Tn5 loading scalebarcodes

DOI

https://dx.doi.org/10.17504/protocols.io.j8nlk1w1wg5r/v1

Koen Theunis^1,2,3,4

¹Laboratory of Computational Biology, VIB Center for AI & Computational Biology, Leuven, Belgium;
²VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium;
³Department of Human Genetics, KU Leuven, Leuven, Belgium;
⁴Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, United States

Aertslab

Koen Theunis

Laboratory of Computational Biology, VIB Center for AI & Com...

DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk1w1wg5r/v1

Protocol Citation: Koen Theunis 2026. Tn5 loading scalebarcodes. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1w1wg5r/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: Working

We use this protocol and it's working

Created: January 09, 2026

Last Modified: January 12, 2026

Protocol Integer ID: 238301

Keywords: ASAPCRN, Tn5, scaleATAC, scATAC-seq, tn5 transposase, scalebarcodes this protocol, barcoded adapter complex, loading scalebarcode, tn5, adapter complex

Funders Acknowledgements:

Aligning Science Across Parkinson’s

Grant ID: ASAP-000430

Aligning Science Across Parkinson’s

Grant ID: ASAP-025179

Abstract

This protocol describes how to prepare barcoded adapter complexes that can be loaded onto Tn5 transposase.

Attachments

Tn5_loading_scalebar...

13KB

Guidelines

All mixes contain glycerol so make sure to pipet slowly and sufficient for proper mixing.

Materials

 
ABCDEF
Oligo
  anneal buffer BStock conc Final conc V (µl)
Tris-HCl (pH 8)1000mM40mM40,0
NaCl5000mM50mM10,0
UltraPure Water    950,0
Total Volume    1000,0
  
ABCD
NameTruseq_R2_SBS12_partialTn5_96plx_idxME
R2_18_ME_scl_4CGTGTGCTCTTCCGATCTCTGCTTCCAGATGTGTATAAGAGACAG
R2_18_ME_scl_5CGTGTGCTCTTCCGATCTGGTCACGAAGATGTGTATAAGAGACAG
R2_18_ME_scl_6CGTGTGCTCTTCCGATCTAACTGTAGAGATGTGTATAAGAGACAG
R2_18_ME_scl_16CGTGTGCTCTTCCGATCTGCGGCACAAGATGTGTATAAGAGACAG
R2_18_ME_scl_17CGTGTGCTCTTCCGATCTCTAGCGCTAGATGTGTATAAGAGACAG
R2_18_ME_scl_18CGTGTGCTCTTCCGATCTTCGATATCAGATGTGTATAAGAGACAG
R2_18_ME_scl_28CGTGTGCTCTTCCGATCTCGCCGATCAGATGTGTATAAGAGACAG
R2_18_ME_scl_29CGTGTGCTCTTCCGATCTTCTCTACTAGATGTGTATAAGAGACAG
R2_18_ME_scl_30CGTGTGCTCTTCCGATCTCTCTCGTCAGATGTGTATAAGAGACAG
R2_18_ME_scl_40CGTGTGCTCTTCCGATCTAACGTTCCAGATGTGTATAAGAGACAG
R2_18_ME_scl_41CGTGTGCTCTTCCGATCTGCAGAATTAGATGTGTATAAGAGACAG
R2_18_ME_scl_42CGTGTGCTCTTCCGATCTATGAGGCCAGATGTGTATAAGAGACAG
R2_18_ME_scl_52CGTGTGCTCTTCCGATCTTGGATCGAAGATGTGTATAAGAGACAG
R2_18_ME_scl_53CGTGTGCTCTTCCGATCTAGTTCAGGAGATGTGTATAAGAGACAG
R2_18_ME_scl_54CGTGTGCTCTTCCGATCTGACCTGAAAGATGTGTATAAGAGACAG
R2_18_ME_scl_64CGTGTGCTCTTCCGATCTGCGCAAGCAGATGTGTATAAGAGACAG
R2_18_ME_scl_65CGTGTGCTCTTCCGATCTAAGATACTAGATGTGTATAAGAGACAG
R2_18_ME_scl_66CGTGTGCTCTTCCGATCTGGAGCGTCAGATGTGTATAAGAGACAG
R2_18_ME_scl_76CGTGTGCTCTTCCGATCTTCCAACGCAGATGTGTATAAGAGACAG
R2_18_ME_scl_77CGTGTGCTCTTCCGATCTCCGTGAAGAGATGTGTATAAGAGACAG
R2_18_ME_scl_78CGTGTGCTCTTCCGATCTTTACAGGAAGATGTGTATAAGAGACAG
R2_18_ME_scl_88CGTGTGCTCTTCCGATCTTAAGGTCAAGATGTGTATAAGAGACAG
R2_18_ME_scl_89CGTGTGCTCTTCCGATCTTTGCCTAGAGATGTGTATAAGAGACAG
R2_18_ME_scl_90CGTGTGCTCTTCCGATCTCCATTCGAAGATGTGTATAAGAGACAG
NameNextera_R1_A14ME
R1-A-oligoTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
NameME'
Nextera_Mosaic_End_REVCOMP/5Phos/CTGTCTCTTATACACATCT
Order HPLC purified oligo's
 
 
ABCDEF
Tn5
  dilution bufferStock conc Final conc V (µl)
Tris-HCl (pH 7.4)1000mM50mM250,0
NaCl5000mM100mM100,0
EDTA50mM0,1mM10,0
DTT1000mM1mM5,0
Glycerol100%50%2500,0
Water    2135,0
Total Volume    5000,0
Add DTT fresh
 

Troubleshooting

Prepare oligo's

Dissolve oligo's in Oligo annealing buffer (40mM Tris-HCl (pH 8), 50mM NaCl)
to a stock of Concentration100 micromolar (µM)  .
Work in 96-well plates for easy pipetting.
 

Anneal oligo's

Mix oligo's equimolar for each barcode: 
 
ABCD
µLStock
A=R13100µM
B=R2-barcodedoligo1-243100µM
ME'6100µM
 

5min 95°C, ramp (-1°C/s) to 65°C, 5min 65°C, ramp (-1°C/s) to 4°C.
 
AB
Temperature
  (°C)Time
95 °C5min
ramp (-1°C/s)
65 °C5min
ramp (-1°C/s)
4 °CHold
 Adapters are now Concentration50 micromolar (µM)  .

Dilute oligo's and Tn5

 
ABCDEF
 Stock conc Final conc V (µl)
oligo mix50µM24mM12,0
Tn5 dil buffer    13,0
Total Volume    25,0
 

 
ABCDE
StorageVol (µL)Conc (ng/µL)Molarity (µM)
EMBL Tn5-8020100018
 
ABCDEF
Tn5
  (diluted in fresh dilution buffer)Stock conc Final conc V (µl)
Tn518µM6µM20,0
Tn5 dil buffer    40,0
Total Volume    60,0
Tn5 is now 333,33 ng/µL; 6 µM

Load Tn5

 
ABCDEF
Tn5 loading on iceStock conc Final conc V (µl)
Tn5 (diluted in fresh dilution
  buffer)6µM5µM5,0
adapter1-2424µM4µM1,0
Total Volume    6,0
Repeat this for every barcoded adapter mix. 
Adapter loaded Tn5 is now max at 4 µM = 222,22ng/µL functional Tn5.

Incubate for Duration00:45:00   @ Temperature23 °C   on a heater/shaker (300 RPM).

Dilute Tn5 to stock concentration and store

Dilute to 62,5 ng/µL for 50 µL reactions
Dilute to 25 ng/µL for 15 µL tagmentation reactions (like 10x Genomics ATAC)

Store at -20 °C.

Protocol references

Our barcoded oligo's are modified versions of the oligonucleotide list that was published in: Mulqueen, R.M., Pokholok, D., O’Connell, B.L. et al. High-content single-cell combinatorial indexing. Nat Biotechnol 39, 1574–1580 (2021). https://doi.org/10.1038/s41587-021-00962-z

DIY Tn5 protocols would not have been possible without the work of: Simone Picelli, Åsa K. Björklund, Björn Reinius, Sven Sagasser, Gösta Winberg and Rickard Sandberg. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. http://www.genome.org/cgi/doi/10.1101/gr.177881.114.

The source of our Tn5 is EMBL and they describe it in the following paper:
Hennig BP, Velten L, Racke I, Tu CS, Thoms M, Rybin V, Besir H, Remans K, Steinmetz LM. Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 (Bethesda). 2018 Jan 4;8(1):79-89. doi: 10.1534/g3.117.300257. PMID: 29118030; PMCID: PMC5765368.

A	B	C	D	E	F
Oligo anneal buffer B	Stock conc		Final conc		V (µl)
Tris-HCl (pH 8)	1000	mM	40	mM	40,0
NaCl	5000	mM	50	mM	10,0
UltraPure Water					950,0
Total Volume					1000,0

A	B	C	D
Name	Truseq_R2_SBS12_partial	Tn5_96plx_idx	ME
R2_18_ME_scl_4	CGTGTGCTCTTCCGATCT	CTGCTTCC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_5	CGTGTGCTCTTCCGATCT	GGTCACGA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_6	CGTGTGCTCTTCCGATCT	AACTGTAG	AGATGTGTATAAGAGACAG
R2_18_ME_scl_16	CGTGTGCTCTTCCGATCT	GCGGCACA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_17	CGTGTGCTCTTCCGATCT	CTAGCGCT	AGATGTGTATAAGAGACAG
R2_18_ME_scl_18	CGTGTGCTCTTCCGATCT	TCGATATC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_28	CGTGTGCTCTTCCGATCT	CGCCGATC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_29	CGTGTGCTCTTCCGATCT	TCTCTACT	AGATGTGTATAAGAGACAG
R2_18_ME_scl_30	CGTGTGCTCTTCCGATCT	CTCTCGTC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_40	CGTGTGCTCTTCCGATCT	AACGTTCC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_41	CGTGTGCTCTTCCGATCT	GCAGAATT	AGATGTGTATAAGAGACAG
R2_18_ME_scl_42	CGTGTGCTCTTCCGATCT	ATGAGGCC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_52	CGTGTGCTCTTCCGATCT	TGGATCGA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_53	CGTGTGCTCTTCCGATCT	AGTTCAGG	AGATGTGTATAAGAGACAG
R2_18_ME_scl_54	CGTGTGCTCTTCCGATCT	GACCTGAA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_64	CGTGTGCTCTTCCGATCT	GCGCAAGC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_65	CGTGTGCTCTTCCGATCT	AAGATACT	AGATGTGTATAAGAGACAG
R2_18_ME_scl_66	CGTGTGCTCTTCCGATCT	GGAGCGTC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_76	CGTGTGCTCTTCCGATCT	TCCAACGC	AGATGTGTATAAGAGACAG
R2_18_ME_scl_77	CGTGTGCTCTTCCGATCT	CCGTGAAG	AGATGTGTATAAGAGACAG
R2_18_ME_scl_78	CGTGTGCTCTTCCGATCT	TTACAGGA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_88	CGTGTGCTCTTCCGATCT	TAAGGTCA	AGATGTGTATAAGAGACAG
R2_18_ME_scl_89	CGTGTGCTCTTCCGATCT	TTGCCTAG	AGATGTGTATAAGAGACAG
R2_18_ME_scl_90	CGTGTGCTCTTCCGATCT	CCATTCGA	AGATGTGTATAAGAGACAG

Name	Nextera_R1_A14		ME
R1-A-oligo	TCGTCGGCAGCGTC		AGATGTGTATAAGAGACAG

Name			ME'
Nextera_Mosaic_End_REVCOMP			/5Phos/CTGTCTCTTATACACATCT

A	B	C	D	E	F
Tn5 dilution buffer	Stock conc		Final conc		V (µl)
Tris-HCl (pH 7.4)	1000	mM	50	mM	250,0
NaCl	5000	mM	100	mM	100,0
EDTA	50	mM	0,1	mM	10,0
DTT	1000	mM	1	mM	5,0
Glycerol	100	%	50	%	2500,0
Water					2135,0
Total Volume					5000,0

A	B	C	D
	µL	Stock
A=R1	3	100	µM
B=R2-barcodedoligo1-24	3	100	µM
ME'	6	100	µM

	A	B
	Temperature (°C)	Time
	95 °C	5min
	ramp (-1°C/s)
	65 °C	5min
	ramp (-1°C/s)
	4 °C	Hold

A	B	C	D	E
	Storage	Vol (µL)	Conc (ng/µL)	Molarity (µM)
EMBL Tn5	-80	20	1000	18

A	B	C	D	E	F
Tn5 (diluted in fresh dilution buffer)	Stock conc		Final conc		V (µl)
Tn5	18	µM	6	µM	20,0
Tn5 dil buffer					40,0
Total Volume					60,0

A	B	C	D	E	F
Tn5 loading on ice	Stock conc		Final conc		V (µl)
Tn5 (diluted in fresh dilution buffer)	6	µM	5	µM	5,0
adapter1-24	24	µM	4	µM	1,0
Total Volume					6,0

Public workspaceTn5 loading scalebarcodes

Tn5 loading scalebarcodes