TITLE: High-throughput sequencing (HTS)  oligos and methods to prepare oligos for HTS applications
AUTHORS: John B. Ridenour, Rafal Donczew

[DESCRIPTION]
In this protocol, we provide sequences of oligos used for high-throughput sequencing (HTS) applications and describe methods for preparing the oligos for HTS applications. This protocol (including oligo sequences and oligo preparation) is based on previously described methods with minor modifications (Foley et al., 2019; Glenn et al., 2019; and Veeranagouda et al., 2019). 

REFERENCES

Foley JW, Zhu C, Jolivet P, Zhu SX, Lu P, Meaney MJ, West RB (2019). Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ. Genome research, 29(11), 1816-1825. https://doi.org/10.1101/gr.234807.118

Glenn TC, Nilsen RA, Kieran TJ, Sanders JG, Bayona-Vásquez NJ, Finger JW, Pierson TW, Bentley KE, Hoffberg SL, Louha S, Garcia-De Leon FJ, Del Rio Portilla MA, Reed KD, Anderson JL, Meece JK, Aggrey SE, Rekaya R, Alabady M, Belanger M, Winker K, Faircloth BC (2019). Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext). PeerJ, 7, e7755. https://doi.org/10.7717/peerj.7755

Veeranagouda Y, Remaury A, Guillemot JC, Didier M (2019). RNA Fragmentation and Sequencing (RF‐Seq): Cost‐Effective, Time‐Efficient, and High‐Throughput 3′ mRNA Sequencing Library Construction in a Single Tube. Current Protocols in Molecular Biology, 129(1). https://doi.org/10.1002/cpmb.109

[GUIDELINES]
Standard laboratory guidelines and practices should be followed when performing this protocol. 

[STEPS]
SECTION: Preparation of Y-yoke adapters
2. Briefly centrifuge the lyophilized adapter oligos at room temperature.
SECTION: Preparation of Y-yoke adapters
3. Prepare a 100 µM stock of each adapter oligo. Add TLEN buffer at a volume 10 times the nmol of the oligo. For example, if the quantity of a given adapter oligo is nmol, add 1265 µl of TLEN buffer.
SECTION: Preparation of Y-yoke adapters
4. Vortex the adapter oligo suspensions on setting 4 for 2 s, incubate at room temperature for 5 min, and briefly centrifuge at room temperature.
SECTION: Preparation of Y-yoke adapters
5. Repeat Step 4 once to ensure adapter oligos are fully resuspended.
SECTION: Preparation of Y-yoke adapters
6. Combine equal volumes of each adapter oligo in a 1.5 ml microcentrifuge tube, vortex on setting 4 for 5 s, and briefly centrifuge at room temperature.

 

SECTION: Preparation of Y-yoke adapters
7. Split the adapter oligo mixture into 50 µl aliquots in 0.2 ml PCR tubes or strips.
SECTION: Preparation of Y-yoke adapters
8. Anneal the adapter oligos in a thermocycler with heated lid using the following conditions: 95°C for 2 min, 73 cycles of cooling (minus 1°C per min) to reach 21°C, and 20°C for 10 min.
SECTION: Preparation of Y-yoke adapters
9. Pool aliquots of annealed adapter oligos (50 µM) in a 1.5 ml screw-cap microcentrifuge tube, vortex on setting 4 for 5 s, and briefly centrifuge at room temperature. This pool is a 50 µM stock of annealed adapters.
SECTION: Preparation of Y-yoke adapters
10. Prepare 15 µM, 1.5 µM, and 0.15 µM stocks of the annealed adapters in TLEN buffer by serial dilution of the 50 µM stock.
SECTION: Preparation of Y-yoke adapters
11. Split the annealed adapter stocks into 50 µl aliquots in 1.5 ml screw-cap microcentrifuge tubes and store at -20°C.

 

SECTION: Preparation of Y-yoke adapters
12. Shortly before use, thaw annealed adapter stocks on ice, vortex on setting 4 for 1 s, and briefly centrifuge at room temperature. Avoid freeze-thaw cycles when possible.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
14. Briefly centrifuge the lyophilized library oligos at room temperature.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
15. Prepare a 100 µM stock of each library oligo. Add TLE buffer at a volume 10 times the nmol of the oligo. For example, if the quantity of a given adapter oligo is nmol, add 590 µl of TLE buffer.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
16. Vortex the library oligo suspensions on setting 4 for 2 s, incubate at room temperature for 5 min, and briefly centrifuge at room temperature.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
17. Repeat Step 16 once to ensure library oligos are fully resuspended.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
18. Prepare 10 µM primer pair mixtures. Combine 10 µl of a given i5 library oligo, 10 µl of a given i7 library oligo, and 180 µl of TLE buffer in a 1.5 ml screw-cap microcentrifuge tube. 

 

SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
19. Vortex 10 µM primer pair mixtures on setting 4 for 5 s and briefly centrifuge at room temperature.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
20. Prepare 5 µM primer pair mixtures. Combine 50 µl of a given 10 µM primer pair mixture and 50 µl of TLE buffer in a 1.5 ml screw-cap microcentrifuge tube.

 

SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
21. Vortex 5 µM primer pair mixtures on setting 4 for 5 s and briefly centrifuge at room temperature.
SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
23. Shortly before use, thaw primer pair mixtures on ice, vortex on setting 4 for 1 s, and briefly centrifuge at room temperature. Avoid freeze-thaw cycles when possible.
SECTION: Preparation of 3´ mRNA capture oligos
24. Briefly centrifuge the lyophilized capture oligos at room temperature.
SECTION: Preparation of 3´ mRNA capture oligos
25. Prepare a 100 µM stock of each capture oligo. Add nuclease-free water at a volume 10 times the nmol of the oligo. For example, if the quantity of a given capture oligo is 1 nmol, add 101 µl of nuclease-free water.
SECTION: Preparation of 3´ mRNA capture oligos
26. Vortex the capture oligo suspensions on setting 4 for 2 s, incubate on ice for 10 min, and briefly centrifuge at room temperature.
SECTION: Preparation of 3´ mRNA capture oligos
27. Repeat Step 26 once to ensure capture oligos are fully resuspended.
SECTION: Preparation of 3´ mRNA capture oligos
28. Prepare 20 µM stocks of the capture oligos in nuclease-free water in 1.5 ml screw-cap microcentrifuge tubes and store at -20°C.

 

SECTION: Preparation of 3´ mRNA capture oligos
29. Shortly before use, thaw capture oligo stocks on ice, vortex on setting 4 for 1 s, and briefly centrifuge at room temperature. Avoid freeze-thaw cycles when possible.
SECTION: Preparation of Y-yoke adapters
1. Prepare the following buffer in a 50 ml conical tube:

TLEN (Tris-HCl, Low EDTA, NaCl) buffer
 

 

SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
13. Prepare the following buffer in a 50 ml conical tube:

TLE (Tris-HCl, Low EDTA) buffer
 

 

SECTION: Strategy for pairing and selecting of library barcoding/enrichment oligos
30. Use the following schematic to select pairs of library barcoding/enrichment oligos to use for a given project.

 

 


SECTION: Preparation of library barcoding/enrichment oligos and mixes of oligo pairs
22. Store primer pair mixtures at -20°C.