TITLE: Yale University_Spatial ATAC Sequencing for Fixed Fresh Frozen Human Lymph node Tissue via DBiT-seq
AUTHORS: Negin Farzad, Yanxiang Deng, Archibald Enninful, yao.lu.yl, Rong Fan

[DESCRIPTION]
This protocol describes the use of Deterministic Barcoding in Tissue for spatial ATAC sequencing to construct an epigenome atlas of fixed fresh frozen human lymph node 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 method spatially resolved genome-wide profiling of  transposase-accessible chromatin sequencing for chromatin accessibility.This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial epigenomic analysis. 

[BEFORE_START]
The protocol below describes the reagents, equipment, and specific experimental steps for using the spatial-ATAC-seq platform on fixed fresh frozen tissue slides. The 20 μm microfluidic channel width were designed and validated to provide spatial epigenomic profiles on human lymph note tissue sections. 

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
3. 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
1. 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
2. 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 Barcodes (A and B).
28. Thoroughly mix the ligation linker 1 with each barcode A1-50 at and ligation linker 2 with each barcode B1-50 at a 1:1 ratio.
SECTION: Preparing Barcodes (A and B).
29. Place the 50 mixes in a thermal cycle and heat to 97˚C to anneal.
SECTION: Preparing Barcodes (A and B).
30. Slowly cool to room temperature at a rate of -0.1˚C/sec.

Store at -20˚C for up to 6 months.
SECTION: Preparing the Tissue Slide
31. 25% sucrose overnight bath.
SECTION: Preparing the Tissue Slide
32. Embed in OCT frozen on dry ice.
SECTION: Preparing the Tissue Slide
33. Section at ~7 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
34. 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
42. Remove stored sections from the freezer and allow to warm to room temperature for 10 minutes.
SECTION: Tissue Preparation
43. Clean sections by pipetting 2 mL of PBS-RI across the tissue.
SECTION: Tissue Preparation
44. If sections are not yet fixed, then fix here with 1 mL 0.2% PFA ,without methanol in 1X PBS,
 by applying on top of the tissue and incubate for 5 min at room temperature.
SECTION: Tissue Preparation
46. Wash the slides with 1 mL of PBS for 1 minute and then dip the slide in a 50 mL falcon tube with DI water.
SECTION: Tissue Preparation
47. Dry the section with gentle air flow.
SECTION: Tissue Preparation
48. Take a full pre-scan image of the section at the desired optical resolution (Figure 4A). 

Recommended 10X resolution.

 
 

SECTION: Tissue Preparation
49. Attach the Antibody PDMS reservoir and 12mm diameter clamp to the tissue.





SECTION: Tissue Preparation
51. Wash the tissue with 500uL wash buffer for 5 min
make sure to prepare wash buffer fresh each time:

Wash Buffer (10 mM Tris-HCl pH 8.0; 10 mM NaCl; 3 mM MgCl2; 1% BSA; 0.1% Tween-20) 




SECTION: Tissue Preparation
52. Prepare the transposition mix with the loaded Tn5 transposon and add 100uL to the tissue.

 make sure to prepare the Tn5 mix fresh each time
     Volume (200 ul/chip)      Volume (300 ul)      Volume (400 ul)      Reagent (50 ul/reaction)        100 ul      150 ul      200 ul      2X TD Buffer (25 ul)        66 ul      99 ul      132 ul      1X PBS (16.5 ul)        2 ul      3 ul      4 ul      10% Tween-20 (final 0.1% v/v) (0.5 ul)        2 ul      3 ul      4 ul      1% Digitonin (final 0.01% v/v) (0.5 ul)        10 ul      15 ul      20 ul      Transposome (2.5 ul)        20 ul      30 ul      40 ul      Nuclease-free H2O (5 ul)   

 


SECTION: Tissue Preparation
53. Incubate the tissue at  37 ºC for 30 min
SECTION: Tissue Preparation
54. Stop the tagmentation reaction by adding 500 μL of 40 mM EDTA onto the tissue and incubating at room temperature for 5 min.




SECTION: Tissue Preparation
None. 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).

 
 

None. Prepare barcode solution. 
None. Introduce final barcode A mixture to the tissue. 
None. Aspirate remaining barcode A mixture from inlets/outlets.
None. Clean the channels by flushing 1X NEB buffer 3.1 with 1% Enzymatics RNase Inhibitor for 10 min.
None. 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
None. Attach Chip-B centered on the tissue. 
SECTION: In-Tissue Ligation
None. Prepare the ligation mixture. 
SECTION: In-Tissue Ligation
None. Prepare barcode B mixture. 
SECTION: In-Tissue Ligation
None. Introduce final barcode B mixture to the tissue. 
SECTION: In-Tissue Ligation
None. Clean the channels with wash buffer for 10 min. 
SECTION: In-Tissue Ligation
None. 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
None. Take a final post-scan of the tissue section (Figure 4B).
 

SECTION: Lysis and Sub-library Generation
None. 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
None. Tightly apply the lysis clamp.
SECTION: Lysis and Sub-library Generation
None. 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
None. 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
None. 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
None. 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
None. 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
None. 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
None. Add 100 µL of resuspended DynabeadsTM MyOneTM Streptavidin C1 magnetic beads to each lysate.
SECTION: cDNA Purification
None. Rotate at 30 RPM and room temperature for 60 min for binding to occur.
SECTION: cDNA Purification
None. Wash beads. 
SECTION: In-Tissue Ligation
None. Scan the channels over the tissue to ensure proper placement (Figure 4D). 
 

SECTION: In-Tissue Ligation
None. Firmly clamp as before.
SECTION: Ampure Purification
83. 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: Tissue Preparation
45. Remove the formaldehyde by pipetting it off and quench the tissue with 1mL 1.25M glycine for 5
min at room temperature.


SECTION: Tissue Preparation
50. Permeabilize the tissue with 500uL 0.1 Xlysis buffer and incubate at room temperature for 15 min

Make sure to prepare 0.1x lysis buffer fresh each time. using the following reagents:
1X Lysis Buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 3
mM MgCl2; 0.1% Tween-20; 0.1% NP-40; 0.01% Digitonin; 1% BSA) 
Dilute down to 0.1X with lysis dilution buffer
(10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 3 mM MgCl2; 1% BSA)


SECTION: Transposome assembly preparation
35. 
 
Prepare the transposome assembly annealing buffer ( The Tn5 annealing buffer contains 400 μl of 1 M Tris-HCl pH 8.0, 100 μl of Nacl 5 M and 9.5 ml water. This annealing buffer is
used to resuspend each oligos of Tn5-Rev, Tn5-A and Tn5-B in this annealing
buffer to stock concentrations of 100 μM.
 
SECTION: Transposome assembly preparation
36. Resuspend the oligos ( Tn5Erev, Tn5ME-A , and Tn5ME-B) in Annealing Buffer to stock
  concentration of 100 µM.
 
SECTION: Transposome assembly preparation
37. In a PCR tube, mix 10 μl of oligo Rev with 10 μl of oligo A.
 
SECTION: Transposome assembly preparation
38. In a separate PCR tube, mix 10 μl of oligo Rev with 10 μl of oligo B   

SECTION: Transposome assembly preparation
39. Vortex the tubes and place them in a thermocycler, and run the thermocycler with the following program:


 
 
     Temperature
        Time        95 °C      5 min        Cool to 65
  C      -0.1C/second        65 °C      5 min        Cool to 4
  °C      -0.1
  C/second   



The Annealed linker oligos can be stored at -20 C.
 
 
SECTION: Transposome assembly preparation
40. Mix 5 μl of each  prepared oligo A/oligo Rev and oligo B/oligo Rev at the ratio of 1:1 in a pcr tube.
 
SECTION: Transposome assembly preparation
41. Add10 μl of unloaded Tn5 transposome (Diagnode Tagmentase) to the mixture of  oligo A/oligo Rev and oligo B/oligo Rev.
After vortexing the mixture briefly, incubate the tube at 23 ºC for 30 minutes in a thermocycler. The product can be use at the same day or stored in -20 ºC with 10 μl glycerol up to 60 days.
SECTION: DBiT-seq assembly and Barcoding
56. Refer to https://www.protocols.io/view/spatial-multi-omics-sequencing-for-fixed-tissue-vi-3byl4j2kolo5/v1
SECTION: Lysis
57. Take a small PDMS solution reservoir and place it on the tissue slide so that the reservoir opening is directly over the barcoded ROI. 
SECTION: Lysis
58. Load the slide into a lysis clamping device and tighten screws as much as possible by hand.
SECTION: Lysis
59. Image the lysis chip on the ROI using a 10x objective lens.
SECTION: Lysis
60. Prepare the reverse crosslinking lysis solution. 




 

 
     Reagent      Volume
        Stock
  Concentration      Final
  Concentration        Tris-HCl (pH 8.0)      7.5 μL      1M      50 mM        EDTA      0.3 μL      0.5M      1 mM        SDS      15 μL      10%      1%        NaCl      6 μL      5M      200 mM        proteinase K      3 μL      20mg/mL      0.4 mg/mL        dH2O      118.2 μL      -      -   


SECTION: Lysis
61. Add approximately 50- 100μL of the reverse crosslinking lysis solution into the lysis chip. CRITICAL STEP Make sure to record the volume added of the lysis solution for the remaining steps.
SECTION: Lysis
62. Cover the ROI hole using parafilm and load the slide into a humidifying chamber.
SECTION: Lysis
63. Incubate tissue the slide with PDMS reservoir and Lysis Solution at 58 ºC for 2 hours to reverse formaldehyde crosslinks.
SECTION: Lysis
64. After incubation, collect the lysate in a 200μL PCR tube (or 1.5mL tube) and seal the tube with parafilm.
SECTION: Lysis
65. Incubate the lysate at 65 ºC with rotation overnight.
SECTION: Ampure Purification
82. Transfer the clear supernatant to a new tube.
SECTION: Ampure Purification
81. Place the tubes back on the magnet and incubate until the liquid is fully clear.
SECTION: Ampure Purification
80. Incubate the tubes at room temperature for at least 10 min to elute the cDNA off the beads.
SECTION: Ampure Purification
79. Remove the tubes from the magnet and resuspend the beads in 15-20 µL of PCR-grade water or elution buffer depending on downstream application.
SECTION: Ampure Purification
78. Repeat steps 70-75.
SECTION: Ampure Purification
77. Carefully remove and discard the ethanol.
SECTION: Ampure Purification
76. Incubate the tubes at room temperature for at least 30 sec.
SECTION: Ampure Purification
75. Carefully remove and discard the supernatant.
SECTION: Ampure Purification
74. Keep the tubes on the magnet and add 200 µL of 80% ethanol.
SECTION: Ampure Purification
73. Mix thoroughly by pipetting.
SECTION: Ampure Purification
72. Perform a 1X SPRI purification by adding 45uL Ampure XP beads to the sample.  Mix thoroughly by pipetting or vortexing. 
SECTION: PCR
69. Prepare the PCR solution:

a.    21uL purified DNA 
b.   2.5uL 25uM Universal or barcoded i5 primer 
c.    2.5uL 25uM Universal or barcoded i7 primer
(using different indices if needed) 
d.   25uL 2X NEBNext Master Mix 
Pipette up and down to mix once you have added everything. 
Briefly vortex and spin down the tubes.
Place the tube in the NEW thermocycler and run the following protocol with a heated
lid:
a.    Lid should be heated at 105°C
b.   Used default ramp rate 
 
     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: Purification and Library Generation
66.  If necessary, transfer the lysate to a 1.5mL microcentrifuge tube.
SECTION: Purification and Library Generation
67.  During this transfer, carefully use a P200 pipette to measure the volume of the lysate, for an accurate PCR product: DNA fragment ratio in the Zymo purification.
SECTION: Purification and Library Generation
68.  Purify the lysate with the Zymo DNA Clean & Concentrator-5 according to the kit protocol:
a. Follow the 5:1 DNA Binding Buffer: DNA Sample
 Use a pipette to accurately determine the initial volume of the PCR Product
b.For the final elution, elute with 100 uL of DNA Elution buffer 


SECTION: PCR
70.      Determine Additional Cycles


1. Prepare preliminary qPCR solution :
Pre-amplified library , 5uL
Pre-amplified solution,0.5uL
25uM Universal or barcoded i5 primer, 0.5uL
25uM Universal or barcoded i7 primer (using different indices if needed), 5uL
NEBNext Master Mix, 3.25uL
Fuclease-free water, 0.75uL
For a final concentration of 1X EvaGreen Dye, 0.75 uL

Pipette up and down to mix once you have added everything.
Briefly vortex and spin down the tubes.
Place the tube in the NEW thermocycler and run the following protocol with a heated lid:
Lid should be heated at 105°C
Use default ramp rate
 
  Denaturation  	  98˚C  	  30 sec  	
  Denaturation  	  98˚C  	  10 sec  	  20 cylces 
  Annealing  	          63˚C  	  30 sec  	
  Extension  	           72˚C  	  1 min

 2.   Use the qPCR data in Excel to determine the cycle where you reach 1/3 of the
saturated signal in qPCR
Graph the qPCR fluorescence values 
It’s a good run if it plateaus in less than 10 cycles 
((fluorescence value at cycle 20 - fluorescence value at cycle 1)/3) + fluorescence value at
cycle 1 = 1/3 Saturated Signal Fluorescence Value
Determine how many cycles it takes to get to this 1/3 Saturated Signal Fluorescence
Value. If in the middle of two cycles, always take the lower.  

Note:Sometimes, all the graphed values may be negative if the amount of DNA Is very
high. This is fine, and just proceed to determine the number of cycles.  

SECTION: Sequencing
84. Send the samples for 100 million reads (30 Gb) and 150 bp paired end sequencing.
SECTION: Data Processing
85. Transfer the two filtered sequences from Step 125 to Cell Ranger ATAC format (10x Genomics)
SECTION: Data Processing
86. Align the filtered reads to the reference genome (e.g., mouse reference (mm10) or human reference (GRCh38) genome)
SECTION: Data Processing
87. Adjust the reads for the Tn5 offset and generate an HTML report with per-base quality scores from which the overall sequencing quality of the library can be inferred. This step provides adjusted reads to correct the Tn5 enzyme’s tendency to insert sequencing adapters from true chromatin accessibility sites at positions offset.
SECTION: Data Processing
88. Perform peak calling and generate the number of Tn5 insertion events and fragments, generating a matrix with spatial barcodes. This step generates a BED-like fragment files for data visualization through Seurat packages.
SECTION: Data Processing
89. Evaluate the molecular mechanism of binding and transposition by the Tn5 transposase dimer by generating fragment size distribution plots.

SECTION: Data Processing
90. The Snakemake workflow management system (v5.28.0) pretreatment pipeline was created and used for preprocessing data, including trimming and alignment, and it is publicly available on GitHub at https://github.com/dyxmvp/Spatial_ATAC-seq.
SECTION: PCR
71. Repeat step 68 PCR protocol to amplify the remaining product with the cycles determined in step 69
SECTION: Tissue Preparation
55. Dip in a 50 mL falcon tube with DI water and gently dry with air flow.