TITLE: Spatial Multi-omics Sequencing for Fixed Tissue via DBiT-seq
AUTHORS: Graham Su, archibald.enninful, Yang Liu, Rong Fan

[DESCRIPTION]
This protocol describes the use of Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq) platform to construct a multi-omics atlas on fixed frozen tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section fixed on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics and spatial analysis of a targeted panel of proteins. 

[BEFORE_START]
The protocol below describes the reagents, equipment, and specific experimental steps for using the DBiT-seq platform on frozen fixed tissue slides. The 10μm, 25μm, 50μm microfluidic channel width were designed and validated to provide spatial profiling at different resolutions.

CRITICAL: Work in an RNase-free environment when the microfluidic device is not on the tissue slide. Use RNaseZapTM or other commercially available cleaner solution and filter-tips. Clean surfaces and gloves with RNaseZapTM.

CRITICAL:Keep reagents on ice at all times.

[STEPS]
SECTION: Fabricating the Silicon Wafer Device Mold
1. Prepare a high-resolution computer-aided-design (CAD) file with the desired microfluidics chip design. A CAD file is also available in the link provided. (https://ars.els-cdn.com/content/image/1-s2.0-S2666166721002392-mmc4.zip)
SECTION: Fabricating the Silicon Wafer Device Mold
2. Prepare chrome photomasks of the microfluidics chip by printing the high-resolution CAD files onto a glass substrate (Figure 1). 

  
NOTE: We have outsourced this step (Front Range Photomask, USA). 

Using these masks, prepare a replica mold as follows:
SECTION: Fabricating the Silicon Wafer Device Mold
3. Start the process by cleaning a 4-inch silicon wafer with 100% acetone (Aldrich) and then 100% isopropanol (Aldrich), then dry with compressed air. 

NOTE: Acetone and isopropanol are mildly toxic. Use proper PPE when handling and discard waste in the appropriate containers.
SECTION: Fabricating the Silicon Wafer Device Mold
4. Bake the wafer at 180°C for 10 min on a hot plate to dry it out.
SECTION: Fabricating the Silicon Wafer Device Mold
5. Use a spin coater to evenly spread SU-8 2010 photoresist (MicroChem) for 10 μm device or SU-8 2025 (MicroChem) for 25 μm and 50 μm device onto the wafer at 500 rpm for 5 seconds followed by 1100 rpm (10 μm device), 3500 rpm (25 μm device) or 1750 rpm (50 μm device) for 40 seconds.
SECTION: Fabricating the Silicon Wafer Device Mold
6. Soft bake the wafer for 3 min at 65°C (25 μm and 50 μm device) and 4 min (10 μm device) or 6 min (25 μm and 50 μm device) at 95°C.
SECTION: Fabricating the Silicon Wafer Device Mold
7. Expose the SU-8 on the wafer through the photomask using Mask Aligner with a dose of 150 mJ/cm2 UV.
SECTION: Fabricating the Silicon Wafer Device Mold
8. For the post exposure bake, bake the wafer for 1 min at 65°C (25 μm and 50 μm device) and 5 min (10 μm and 25 μm device) or 6 min (50 μm device) at 95°C.
SECTION: Fabricating the Silicon Wafer Device Mold
9. Develop the SU-8 for 4 min (10 μm and 25 μm device) or 5 min (50 μm device) in a bath of ∼50 mL SU-8 developer (MicroChem).
SECTION: Fabricating the Silicon Wafer Device Mold
10. Rinse the wafer with 100% isopropanol and dry with compressed air.
SECTION: Fabricating the Silicon Wafer Device Mold
11. Perform a hard bake by baking the wafer for 10 min at 180°C. 

CRITICAL: This process needs to be carried out in a microelectronics cleanroom. 

Pause Point: The replica mold can now be stored and reused indefinitely.
SECTION: Creating the Acrylic Clamps
12. Prepare the pattern and dimensions for the barcoding clamp and lysis clamp.
SECTION: Creating the Acrylic Clamps
13. Peel covering of acrylic sheet and place it in the laser cutting machine.
SECTION: Creating the Acrylic Clamps
14. Select the program dimensions and cut two pieces for the top and bottom of the barcoding clamp.
SECTION: Creating the Acrylic Clamps
15. Place the top blank piece into the laser cutter and select the pattern to cut out the four holes in the corner for the screws and nuts.
SECTION: Creating the Acrylic Clamps
16. Remove cut-out scraps with a pipet tip or a similar pointed tool.
SECTION: Creating the Acrylic Clamps
17. Repeat steps 15 and 16 for the bottom blank piece.
SECTION: Creating the Acrylic Clamps
18. Repeat steps 13 to 17 for the lysis clamp using the correct dimensions and pattern with an additional hole in the center of the top piece (Example shown in Figure 2). 

   

NOTE: The acrylic clamps can be reused indefinitely.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
19. Thoroughly mix polydimethylsiloxane (PDMS) elastomer base and curing agent (these come together) at a 10:1 ratio (See Figure 3).

  

SECTION: Preparing the Microfluidic Device and reagent reservoirs
20. Pour the mixture into the silicon device mold. 

NOTE: Aim for a chip height of about 5mm.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
21. Place in a vacuum desiccator until all bubbles dissipate from the mixture about 30-60 min.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
22. Cure in an oven at 65-70˚C for a minimum of 2 hours and up to overnight. 

NOTE: Make sure to place on an even surface to prevent uneven curing!
SECTION: Preparing the Microfluidic Device and reagent reservoirs
23. Cut out the cured device and hole punch each of the 50 inlets and outlets for the channels. 

NOTE: Cut both Chip-A and Chip-B in the approximate size of a glass slide. 
NOTE: Make sure there are no PDMS pieces left in the inlets or outlets.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
24. Thoroughly clean the surface of the device with scotch tape.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
25. Pour mixture in a container large enough to cut out two roughly 25x25x5mm PDMS pieces.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
26. For the barcoding reservoir, cut out a 20x20mm piece in the center, barely large enough to surround the inlets of the chips when placed above.
SECTION: Preparing the Microfluidic Device and reagent reservoirs
27. For the lysis reservoir, depending on the device size used, cut out the center to barely surround the barcoded region of the tissue (1x1mm for the 10µm channel device, 2.5x2.5mm for the 25µm channel device). 

NOTE: The reservoirs are reusable. Thoroughly wash with 70% ethanol after each use.  

CRITICAL: Be careful when cutting out the Chips A and B. Make sure not to cut across any patterned areas of the silicon wafer mold, otherwise it will need to be replaced and re-fabricated.
SECTION: Preparing Barcode B
28. Thoroughly mix the ligation linker with each barcode B1-50 at a 1:1 ratio.
SECTION: Preparing Barcode B
29. Place the 50 mixes in a thermal cycle and heat to 97˚C to anneal.
SECTION: Preparing Barcode B
30. Slowly cool to room temperature at a rate of -0.1˚C/sec.
SECTION: Preparing Barcode B
31. Store at -20˚C for up to 6 months.
SECTION: Preparing the Tissue Slide
32. Fix in 4% paraformaldehyde (PFA) in PBS. 

NOTE: PFA is moderately toxic and should be handled in a chemical fume hood with proper PPE. It should be disposed of in the proper waste container. This step is optional.
SECTION: Preparing the Tissue Slide
33. 25% sucrose overnight bath.
SECTION: Preparing the Tissue Slide
34. Embed in OCT frozen on dry ice.
SECTION: Preparing the Tissue Slide
35. Section at ~10 µm thickness for the 25µm channel device and ~5 µm thickness for the 10 µm channel device onto Poly-L-lysine slides.
SECTION: Preparing the Tissue Slide
36. Store slides at -80˚C for up to 6 months until use.  

CRITICAL: Ensure that the tissue is sectioned as evenly as possible, otherwise the risk of cross-flow between the microfluidic channels will be increased. Tissue section should be placed at the center of a glass slide to the greatest extent.
SECTION: Tissue Preparation
37. Remove stored sections from the freezer and allow to warm to room temperature for 10 minutes.
SECTION: Tissue Preparation
38. Clean sections by pipetting 2 mL of PBS-RI across the tissue.
SECTION: Tissue Preparation
39. If sections are not yet fixed, then fix here with 1 mL 4% PFA by applying on top of the tissue and incubate for 20 min at room temperature.
SECTION: Tissue Preparation
40. Wash the slides 3 times with 1 mL of PBS and then dip the slide in a 50 mL falcon tube with DI water.
SECTION: Tissue Preparation
41. Dry the section with gentle air flow.
SECTION: Tissue Preparation
42. Take a full pre-scan image of the section at the desired optical resolution (Figure 4A). 

Recommended 10X resolution.

   

SECTION: Tissue Preparation
43. Block the tissue slide with 1% BSA and 1% RNase Inhibitor in 1X PBS for 30 minutes at 4˚C.
SECTION: Tissue Preparation
44. Remove the blocking buffer and briefly airdry.
SECTION: Tissue Preparation
45. Add a cocktail of the desired DNA-antibody conjugates + blocking buffer and incubate for 30 minutes at 4˚C.
SECTION: Tissue Preparation
46. Wash the slide with 1X PBS three times.
SECTION: Tissue Preparation
47. Dip in a 50 mL falcon tube with DI water and gently dry with air flow.
SECTION: Tissue Preparation
48. Attach Chip-A as shown in Figure 5 centered on the tissue and take another scan of the channels laying on the tissue (Figure 4C).

  
  

SECTION: Tissue Preparation
49. Permeabilize the tissue section.

CRITICAL: These steps should be done in an RNA-free area.
SECTION: In-Tissue Reverse Transcription
50. Prepare the RT mixture. 
SECTION: In-Tissue Reverse Transcription
51. Prepare barcode solution. 
SECTION: In-Tissue Reverse Transcription
52. Introduce final barcode A mixture to the tissue. 
SECTION: In-Tissue Reverse Transcription
53. Aspirate remaining barcode A mixture from inlets/outlets.
SECTION: In-Tissue Reverse Transcription
54. Clean the channels by flushing 1X NEB buffer 3.1 with 1% Enzymatics RNase Inhibitor for 10 min.
SECTION: In-Tissue Reverse Transcription
55. Peel off Chip-A and briefly dip the slide in a 50mL falcon tube with DI water and gently dry with air flow.
SECTION: In-Tissue Ligation
56. Attach Chip-B centered on the tissue. 
SECTION: In-Tissue Ligation
57. Prepare the ligation mixture. 
SECTION: In-Tissue Ligation
58. Prepare barcode B mixture. 
SECTION: In-Tissue Ligation
59. Introduce final barcode B mixture to the tissue. 
SECTION: In-Tissue Ligation
60. Clean the channels with wash buffer for 10 min. 
SECTION: In-Tissue Ligation
61. Peel off Chip-B and dip the slide into a 50mL falcon tube with DI water and gently dry with air flow.
SECTION: In-Tissue Ligation
62. Take a final post-scan of the tissue section (Figure 4B).
  

SECTION: Lysis and Sub-library Generation
63. Place the lysis reservoir directly around the barcoded tissue region. 

NOTE: Try to cover as little extra tissue as possible.
SECTION: Lysis and Sub-library Generation
64. Tightly apply the lysis clamp.
SECTION: Lysis and Sub-library Generation
65. Add lysis solution to lysis reservoir depending on channel dimensions (~20 µL for the 25 µm channels, ~10 µL for the 10 µm channels). 
SECTION: Lysis and Sub-library Generation
66. Place in the humidified chamber and tightly wrap the chamber with parafilm and incubate at 55˚C for 2 hours.
SECTION: Lysis and Sub-library Generation
67. Collect the lysate into a 1.5 mL centrifuge tube. Add the same amount of extra lysis solution to wash out the reservoir and retrieve as much lysate as possible.
SECTION: Lysis and Sub-library Generation
68. Immediately store at -80˚C until next step.  

Pause Point: Stop here for the first day. The lysate should be stable at -80˚C for up to 2 weeks. The Methods Video S1 shows up to this step.
 

SECTION: cDNA Purification
69. The cDNA is purified and bound to Dynabeads™ MyOne™ Streptavidin C1 beads in this step.

Prepare 40 µL DynabeadsTM MyOneTM Streptavidin C1 beads (Thermofisher) per sub-library. 
SECTION: cDNA Purification
70. Purify lysate following the DNA Clean & Concentrator with Zymo-spin IC Columns (RPI Research Products) protocol. Use 100 µL water to elute DNA.
SECTION: cDNA Purification
71. Add 100 µL of resuspended DynabeadsTM MyOneTM Streptavidin C1 magnetic beads to each lysate.
SECTION: cDNA Purification
72. Rotate at 30 RPM and room temperature for 60 min for binding to occur.
SECTION: cDNA Purification
73. Wash beads. 
SECTION: Template Switch
74. This step performs template switching and adds the second PCR handle.

Resuspend the streptavidin beads bound with cDNA in solution containing 44 µL of 5X Maxima RT buffer (ThermoFisher), 44  µL of 20% Ficoll PM-400 solution, 22 µL of 10 mM dNTPs each (ThermoFisher), 5.5 µL RNase Inhibitor (Enzymatics), 11 µL of Maxima H Minus Reverse Transcriptase (ThermoFisher), 5.5 µL of 100 µM of template switch primer, and 88 µL of RNase-free water.
SECTION: Template Switch
75. Rotate beads at 30 RPM and room temperature for 30 min followed by rotation at 42˚C for 90 min.
SECTION: In-Tissue Ligation
56.1. Scan the channels over the tissue to ensure proper placement (Figure 4D). 
  

SECTION: In-Tissue Ligation
56.2. Firmly clamp as before.
SECTION: Tissue Preparation
49.1. Firmly attach the barcoding clamp across the center of the device & slide.
SECTION: Tissue Preparation
49.2. Place the inlet reservoir above the inlets.
SECTION: Tissue Preparation
49.3. Add ~1 mL 0.5% Triton X-100 in PBS to the reservoir
Use a 10 µL pipet tip to pipet up and down inside the inlets to remove bubbles.
SECTION: Tissue Preparation
49.4. Attach the vacuum head to the outlets as shown in Figure 6 and pull the solution through the 50 channels.

NOTE: Make sure all channels are filled with solution.

   

SECTION: Tissue Preparation
49.5. Stop the vacuum and incubate for 20 min at room temperature.
SECTION: Tissue Preparation
49.6. Remove remaining permeabilization solution from the reservoir and the inlets/outlets.
SECTION: Tissue Preparation
49.7. Add ~1 mL 0.5X PBS-RI to the reservoir and flow for about 10 min to wash and clean the channels of Chip-A.
SECTION: In-Tissue Reverse Transcription
50.1. For one experiment, prepare 50 µL of 5X RT Buffer, 32.8 µL of RNase-free water, 1.6 µL Enzymatics RNase inhibitor, 3.2 µL SUPERase• InTM RNase Inhibitor, 12.5 µL of 10 mM dNTPs each, 25 µL of Maxima H Minus Reverse Transcriptase, and 100 µL 0.5X PBS-RI.
SECTION: In-Tissue Reverse Transcription
51.1. In a 96-well PCR plate, add 4 µL of the RT mixture in each of 50 wells. 
SECTION: In-Tissue Reverse Transcription
51.2. Add 1 µL of each 25 µM Barcode A1-50 to the wells and mix thoroughly.
SECTION: In-Tissue Reverse Transcription
52.1. Pipet each barcode A mixture directly into the inlets following the sequence as indicated in Figure 7. This sequence corresponds to the ordering of the channels across the center region of interest (for example, channels from top to bottom of Chip A correspond to 1-50 in the pipetting order).   

SECTION: In-Tissue Reverse Transcription
52.2. Apply the vacuum and pull the solution until all channels are full. 

NOTE: May take as short as 10 sec or as long as 2 min depending on channel dimensions (smaller channels may take longer to fill). 
SECTION: In-Tissue Reverse Transcription
52.3. Incubate in the humidified chamber at room temperature for 30 min followed by 42˚C for 90 min for in-cell reverse transcription (Figure 8).   

SECTION: In-Tissue Ligation
57.1. For one experiment, prepare 69.5 µL RNase-free water, 27 µL 10X T4 Ligation buffer (NEB), 11 µL T4 DNA Ligase (400 U/µL, NEB), 2.2 µL RNase inhibitor (40 U/µL, Enzymatics), 0.7 µL SUPERase• InTM RNase Inhibitor (20 U/µL, Ambion), and 5.4 µL of 5% Triton X-100. Mix with 115.8 µL 1X NEB buffer 3.1 with 1% RNase Inhibitor (Enzymatics).
SECTION: In-Tissue Ligation
58.1. In a 96-well PCR plate, add 4 µL of the ligation mixture in each of 50 wells. 
SECTION: In-Tissue Ligation
58.2. Add 1 µL of each 25 µM Barcode B1-50 to the wells and mix thoroughly.
SECTION: In-Tissue Ligation
59.1. Pipet each barcode B mixture directly into the inlets. 
SECTION: In-Tissue Ligation
59.2. Apply the vacuum and pull the solution until all channels are full. May take as short as 10 sec or as long as 2 min. 

CRITICAL: Do not over vacuum! Make sure the inlets do not run out of barcode B mixture which will cause the channels to lose the mixture and fill with air. 
SECTION: In-Tissue Ligation
59.3. Incubate in the humidified chamber at 37˚C for 30 min for in-cell ligation of barcode B (Figure 8).
SECTION: In-Tissue Ligation
60.1. Prepare wash buffer by mixing 4 mL of 1X PBS, 40 µL of 10% Triton X-100, and 10 µL of SUPERase• InTM RNase Inhibitor.
SECTION: Lysis and Sub-library Generation
65.1. Lysis solution is made with 50 µL 1X PBS, 50 µL of 2X lysis buffer, and 10 µL of proteinase K solution (20mg/mL). 
SECTION: Lysis and Sub-library Generation
65.2. 2X lysis buffer is made up of 20 mM Tris (pH 8.0), 400 mM NaCl, 100 mM EDTA (pH 8.0), and 4.4% SDS.
SECTION: cDNA Purification
69.1. Wash 3 times with 800 µL of 1X B&W buffer with 0.05% Tween-20. 
SECTION: cDNA Purification
69.2. Resuspend beads in 100 µL 2X B&W buffer + 2 µL SUPERase• InTM RNase Inhibitor.
SECTION: cDNA Purification
73.1. Wash twice with 400 µL of 1X B&W buffer with 5 min rotation after resuspending beads. 
SECTION: cDNA Purification
73.2. Wash once more with 400 µL 10 mM Tris containing 0.1% Tween-20 with 5 min rotation after resuspension.
SECTION: PCR
76. Wash beads and resuspend.
SECTION: PCR
76.1. Wash once with 400 µL of 10 mM Tris and 0.1% Tween-20 solution and once more with 400 µL of RNase-free water.
SECTION: PCR
76.2. Resuspend in the PCR mix solution containing 110 µL of 2X Kapa HiFi HotStart Master Mix (Kapa Biosystems), 8.8 µL each of 10 µM stocks of primers 1 and 2, and 92.4 µL of RNase-free water. Transfer 200 µL to four PCR tubes with 50 µL in each.
SECTION: PCR
77. Perform PCR to detach cDNA from beads. 
 
     PCR   Cycling Conditions        Steps      Temperature      Time      Cycles        Initial   Denaturation      95˚C      3 min      1        Denaturation      98˚C      20 sec      5   cycles        Annealing      65˚C      45 sec        Extension      72˚C      3 min   

SECTION: PCR
78. Remove the DynabeadsTM from the PCR solution using a magnetic tube holder.
SECTION: PCR
79. Add Evagreen (Biotium) at a 1X concentration. 

NOTE: This step is optional.
SECTION: PCR
80. Perform PCR again with the following thermocycling conditions. Cycling can also be halted once the qPCR signal begins to plateau. 
     Initial   Denaturation      95˚C      3 min      1        Denaturation      98˚C      20 sec      15   cycles        Annealing      65˚C      20 sec        Extension      72˚C      3 min        Final   Extension      72˚C      5 min      1        Hold      4˚C      forever   
 
SECTION: cDNA Purification
81. Add 80 µL KAPA pure beads for 100 µL of the cDNA sample. 

NOTE: Ensure that beads have been equilibrated to room temperature and are fully resuspended before use.
SECTION: cDNA Purification
82. Mix thoroughly by pipetting.
SECTION: cDNA Purification
83. Incubate the tubes at room temperature for at least 10 min to bind the cDNA to the beads.
SECTION: cDNA Purification
84. Place the tubes on a magnet to capture the beads and incubate until the liquid is clear.
SECTION: cDNA Purification
85. Carefully remove and discard the supernatant.
SECTION: cDNA Purification
86. Keep the tubes on the magnet and add 200 µL of 80% ethanol.
SECTION: cDNA Purification
87. Incubate the tubes at room temperature for at least 30 sec.
SECTION: cDNA Purification
88. Carefully remove and discard the ethanol.
SECTION: cDNA Purification
89. Repeat steps 86-88.
SECTION: cDNA Purification
90. Try to remove all residual ethanol and dry the beads at room temperature for 3-5 min or until all the ethanol has evaporated. 

CRITICAL: Over-drying may result in reduced yield.
SECTION: cDNA Purification
91. Remove the tubes from the magnet and resuspend the beads in 15 µL of PCR-grade water or elution buffer depending on downstream application.
SECTION: cDNA Purification
92. Incubate the tubes at room temperature for at least 10 min to elute the cDNA off the beads.
SECTION: cDNA Purification
93. Place the tubes back on the magnet and incubate until the liquid is fully clear.
SECTION: cDNA Purification
94. Transfer the clear supernatant to a new tube.
SECTION: cDNA Purification
95. Perform Bioanalyzer QC to obtain cDNA length profile and concentration.

Pause Point: Stop here for the second day. Store at 4˚C for 1-2 weeks or up to 6 months at -20˚C.
SECTION: Library Preparation
96. Dilute 0.75 to 1 ng of purified cDNA in PCR-grade water to a total of 5 µL.
SECTION: Library Preparation
97. Add 10 µL of Tagment DNA buffer and 5 µL of Amplicon Tagment Mix to the cDNA for a total of 20 µL.
SECTION: Library Preparation
98. Mix thoroughly and incubate at 55˚C for 5 min.
SECTION: Library Preparation
99. Add and mix 5 µL of NT Buffer to the solution and incubate at room temperature for 5 min.
SECTION: Library Preparation
100. In this order, add 15 µL of the PCR master mix, 8 µL of water, and 1 µL of each 10 µM primer (P5 primer and indexed P7 primer) to the mix for a total of 50 µL.
SECTION: Library Preparation
101. Perform PCR with the following thermocycling conditions.  
     Initial   Denaturation      95˚C      30 sec      1        Denaturation      95˚C      10 sec      12   cycles        Annealing      55˚C      30 sec        Extension      72˚C      30 sec        Final   Extension      72˚C      5 min      1        Hold      4˚C      forever   
 
SECTION: Library Preparation
102. Purify the resulting PCR reaction mix at a 0.7X ratio of KAPA pure beads according to the manufacturer’s manual to generate an Illumina-compatible sequencing library (as in steps 81-95).
SECTION: Library Preparation
103. Perform quality control analysis (Bioanalyzer) to obtain more accurate concentration and yield to prepare for sequencing.