Make consumables and reagents

• 1: Sequencing substrate preparation
• 2: Reservoir assembly station
• 3: Sequencing reservoir assembly
• 4: Linear DISCS preparation
• 5: One pot sequencing buffer

The Sequencing Reservoir is composed of a quartz sequencing substrate, a top reservoir piece, and bottom reservoir piece. The top and bottom pieces are made of polycarbonate with an over-molded TPE gasket that comes into contact with the quartz sequencing substrate. The bottom gasket allows the objective lens and camera sensor to capture images of sequencing in real-time as Lightning Terminators™ are incorporated on the top side of the sequencing substrate. The top piece of the sequencing reservoir forms the sequencing reaction vessel, and the top gasket seals the sequencing substrate to prevent leaks. The area bounded by the top gasket is covered in surface-bound forward and reverse primers. There is no exchange of fluid or cycling required for our truly one pot sequencing.

1 - Sequencing substrate preparation

Introduction

This protocol details materials and methods used to clean and activate quartz substrates for Sequencing Reservoirs.

The quartz substrates go through three stages of cleaning prior to activation: solvent, alkaline, and then an acid clean. Next, the clean slides are activated with piranha acid.

Commercially available MCP4 polymer is deposited on the surface of the activated substrate to provide a three dimensional layer of active NHS groups for oligonucleotide attachment.

Oligonucleotides with 5’ amine modifications are coupled to the slide surface. After blocking, washing, and drying the slides, they are ready to be assembled into Sequencing Reservoirs.

Revision History

Materials and Equipment

Notes Before Beginning

Procedure

1. Clean Z-Cut Quartz Slides

1. Solvent clean
   1. Submerge slides vertically in a bath of semiconductor grade acetone
      • Sonicate 5 minutes
   2. Transfer slides to a bath of semiconductor grade isopropyl alcohol
      • Sonicate 5 minutes
   3. Transfer slides to a bath of methanol
      • Sonicate 5 minutes
2. Alkaline clean
   1. Transfer slides to a bath of 10% potassium hydroxide in methanol (% w/v)
      • Sonicate 20 minutes at 50º C
   2. Transfer slides to a bath of reagent alcohol
      • Sonicate for 20 minutes at 50º C
   3. Repeat steps 1.2.1 through 1.2.2 for a total of two potassium hydroxide and two reagent alcohol washes
   4. Transfer slides to a water bath
      • Sonicate for 5 minutes
   5. Transfer slides to a new water bath
   6. Repeat steps 1.2.4 through 1.2.5 for a total of 3 washes with sonication
   7. Transfer slides to a methanol bath
      • Sonicate for 5 minutes
3. Acid clean
   1. Transfer slides into a fresh bath containing a solution of 50% hydrochloric acid in methanol
      • Sonicate for 30 minutes at 60º C
   2. Transfer slides to a water bath
      • Sonicate for 5 min.
   3. Repeat step 1.3.2 two more times for a total of three washes, using a fresh water bath for each wash
   4. Dry slides thoroughly with filtered compressed nitrogen
      • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface
   5. Store until ready to proceed to acid cleaning step

2. Piranha activate clean slides

1. Transfer slides to a bath containing piranha acid
   • Prepare piranha acid by combining 1 volume 30% hydrogen peroxide to 3 volumes of 5 M sulfuric acid
   • Sonicate for 60 min at 60º C.
2. Transfer slides into a water bath
   • Sonicate for 5 min.
3. Repeat step 2.2 four times for a total of 5 water washes with sonication, using a fresh water bath each time
   • Ensure all traces of piranha acid are removed
4. Dry slides thoroughly with filtered compressed nitrogen
   • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface

3. MCP4 polymer deposition

1. Prepare MCP4 working solution by combining the following reagents:

   • Solution	Final Concentration	Volume for 1 mL
     Nuclease free water		780 – 796 µL
     5x Coat On Coating Solution	1x	200 µL
     50x MCP4 Polymer Concentrate	0.2 – 1x Determine ideal concentration experimentally	4 – 20 µL

   • Note: DO NOT add polymer to solution until slides are ready for incubation
2. Incubate activated slides in MCP4 Working Solution for 30 minutes
   • Slides can be submerged or assembled in fixtures that allow for a pool of working solution to cover a desired area
3. After incubation, slides need to be aggressively washed in deionized water
   • Submerge in running 5 minutes, or submerge in multiple passive baths of deionized water. If slides are assembled in fixtures, recommend disassembling and washing an addition 1-3 minutes vertically in running water.
4. Dry slides thoroughly with filtered compressed nitrogen
   • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface
5. Cure dry slides at 80º C under high vacuum

4. Oligo immobilization

1. Prepare a 250 uM each solution of forward and reverse surface primers in nuclease free water
   • Concentrations can be optimized experimentally
2. Determine ideal spotting conditions for your DNA microarray printer
3. Surface primer solution can be diluted 1:1 with Spot on Spotting Solution when ready to spot primers onto surface
4. Incubate slides overnight

5. Blocking, washing, drying

1. Prepare 1x Block on Blocking solution by diluting stock 1:1 in nuclease free water
2. Fill vertical reaction chambers with 270-300 uL of solution
3. Transfer slides from overnight incubation into the pre-filled reaction chamber
4. Incubate at 50 C for 30 minutes
5. Place slides vertically in a deionized water bath
   1. Incubate 5 min in running water
6. Transfer slides to a 2x SSC + 0.1% SDS solution and incubate 10 minutes with enough shaking to agitate the solution without dislodging slides
7. Place slides vertically in a deionized water bath
   1. Incubate 5 min in running water
8. Dry slides thoroughly with filtered compressed nitrogen
   • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface

2 - Reservoir assembly station

Components

Procedure

3 - Sequencing reservoir assembly

Introduction

This protocol details materials and methods to assemble Sequencing Reservoirs. The reservoir is composed of three major parts: reservoir Top, reservoir Bottom, and sealed in between the two, the sequencing substrate coated in immobilized surface primers.

A vacuum fixture is used to hold the reservoir bottom in place as the sequencing substrate is aligned on top of the gasket of the reservoir bottom. Once well aligned, the vacuum is turned on and will hold the slide firmly to the gasket. The gasketed surface of the reservoir top is then placed on top of the slide, and force is applied to lock the reservoir top legs into the reservoir bottom. Looking down into the reservoir through the top reveals about 20 mm² of the sequencing substrate. The top surface of the substrate is covered in immobilized surface primers and ready for sequencing library hybridization.

A press is used to compress the whole assembled sequencing reservoir to help ensure there are no leaks during subsequent steps.

Revision History

Materials and Equipment

Notes Before Beginning

Sequencing substrates and reservoir tops and bottoms should remain particle free. Components should be stored in clean air tight containers when not in use. For manipulation it is recommended to work in a clean room and/or within a laminar flow hood. All materials for reservoir assembly should be placed within clean space before starting procedure.

Procedure

1. Assemble Reservoir

1. Place reservoir bottom onto the vacuum block with the TPU gasket facing up and the elevated legs in the top left and bottom right corners
2. Using tweezers, carefully place the slide onto the bottom part of the reservoir with the oligo coated side facing up
3. Use tweezers to gently manipulate slide so it is centered over the reservoir bottom
4. Turn the vacuum on so the slide and bottom reservoir are help in place for next steps. Ensure slide is still centered over reservoir bottom
5. Screw on a cap to the reservoir top
6. Hold the reservoir top over the slide and reservoir bottom so the longer legs are positioned over the longer leg inserts of the reservoir bottom
   • Longer pieces of both parts should be at the top left and bottom right corners
7. Once properly positioned, carefully lower reservoir top onto reservoir bottom so the legs are resting on the leg inserts
8. Depress forcefully to finish assembling the reservoir
   • Listen for two audible “clicks” as the reservoir top is fitted into the reservoir bottom

2. Check assembly

1. Turn off vacuum holding reservoir in place and remove assembled reservoir
2. Inspect edges of glass slide to ensure it is still well centered between top and bottom pieces and there are no corners or sides protruding
3. Inspect gasket to slide contact of top and bottom pieces ensures there are no gaps between glass and gaskets

3. Press assembled reservoir to prevent leaking

1. Place reservoir cap-down in reservoir press fixture
2. Turn on press and wait 5 seconds
3. Turn off press and remove reservoir
4. Label reservoir for your experiment

4 - Linear DISCS preparation

Introduction

454’s One Pot Sequencing relies on the 365nm UV TIRF to deblock Lightning Terminator dye linked terminator groups. Stray UV light also deblocks the free Lightning Terminators™ in the bulk solution. The resulting deprotected dNTP competes with LTs during incorporation by Therminator and causes significant leading.

We have developed DISCS (Dark Base In-Situ Cleanup System): which includes Bst 3.0 DNA polymerase from NEB and a set of duplex oligos to quickly consume the deblocked dNTP (dark bases). DISCS is crucial for the success of One Pot Sequencing.

This protocol describes the procedure to prepare linear DISCS for one pot sequencing.

Revision History

Materials and Equipment

Procedure

1. Resuspend fresh oligo pellets from IDT with IDTE to 200 uM stock concentration.
2. Open the linear DISCS worksheet.
3. Enter the required information and the intended volume for each DISCS.
4. Prepare four DISCS individually by mixing 10x ThermoPol buffer, forward oligo, reserve oligo and nuclease free water following the worksheet.
5. Aliquot the mix to 200 uL PCR strip tubes with no more than 50 uL into each tube.
6. Anneal forward and reverse oligos together by a step-down annealing protocol:
   1. 90°C for 60 seconds.
   2. 75°C for 30 seconds.
   3. 70°C for 30 seconds.
   4. 60°C for 30 seconds.
   5. 50°C for 30 seconds.
   6. 40°C for 30 seconds.
   7. Store at 10°
7. Mix all solutions together equally and mix well.
8. Aliquot 20 uL to each tube and store at -20°

5 - One pot sequencing buffer

Introduction

This protocol describes the procedure to prepare one pot sequencing buffer.

Revision History

Materials and Equipment

Procedure

1. Open the sequencing buffer worksheet.
2. Enter the required information.
3. Bring 10X ThermoPol buffer, 100 mM MgSO₄ and DTT from the -20°C freezer to room temperature for 30 minutes to thaw
4. Bring all other chemicals to a clean area.
5. Use a clean container (for example, a glass beaker) to prepare the buffer.
6. Add the required volume of each liquid component to the container.
7. Record the lot information and the quantity added to the worksheet.
8. Make sure the DTT bottle reaches room temperature and there is no condensation on the bottle before opening the cap.
9. Return DTT to the freezer once finished.
10. After all chemicals are added, fill the container with the nuclease free water to 1 mL less than the total volume.
11. Stir slowly to mix.
12. The pH should be around 8.3-8.4. Adjust pH to 8.8 at 25°C with 1 M NaOH.
13. Add nuclease free water to bring mixture up to total volume.
14. Filter the buffer with 0.2 µm filter.
15. Aliquot 1.5 mL buffer to 2.0 mL clean centrifuge tubes for storage at -20°