Jan 14, 2025
Yale University Co_Profile Spatial-ATAC and RNA Sequencing via Deterministic Barcoding in Fresh Frozen Human Lymph Node Tissue
- Negin Farzad1,
- Archibald Enninful1,
- Di Zhang1,
- Rong Fan1
- 1Yale University
- Cellular Senescence Network (SenNet) Method Development Community
External link: https://www.nature.com/articles/s41586-022-05094
Protocol Citation: Negin Farzad, Archibald Enninful, Di Zhang, Rong Fan 2025. Yale University Co_Profile Spatial-ATAC and RNA Sequencing via Deterministic Barcoding in Fresh Frozen Human Lymph Node Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wrezvo5/v1
Manuscript citation:
Deng, Y., Bartosovic, M., Ma, S. et al. Spatial profiling of chromatin accessibility in mouse and human tissues. Nature 609, 375–383 (2022). https://doi.org/10.1038/s41586-022-05094-1
Farzad N, Enninful A, Bao S, Zhang D, Deng Y, Fan R. Spatially resolved epigenome sequencing via Tn5 transposition and deterministic DNA barcoding in tissue. Nat Protoc. 2024 Jun 28. doi: 10.1038/s41596-024-01013-y. Epub ahead of print. PMID: 38943021.
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 10, 2025
Last Modified: January 14, 2025
Protocol Integer ID: 118089
Keywords: epigenome profiling of cellular senescence, fixed fresh frozen human lymph node tissue sample, fresh frozen human lymph node tissue sample, transcriptome atlas, epigenome profiling, fresh frozen human lymph node tissue, cellular senescence, cell resolution, tissue for spatial atac, rna, single cell
Funders Acknowledgements:
Yale TMC for Cellular Senescence in Lymphoid Organs
Grant ID: 1U54AG076043-01
Abstract
This protocol describes the use of Deterministic Barcoding in Tissue for spatial ATAC and RNA sequencing to construct epigenome and transcriptome atlases 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 using DBiT-seq device. This method spatially resolved transcriptome and epigenome profiling of cellular senescence in fresh frozen human lymph node tissue samples.This technique does not directly resolve single cells but can achieve a near-single-cell resolution.
Materials
KEY RESOURCES TABLE
| A | B | C | |
| Name | Catalog number | Vender | |
| Formaldehyde solution | PI28906 | Thermo Fisher Scientific | |
| Glycine | 50046 | Sigma-Aldrich | |
| NaCl | AM9760G | Thermo Fisher Scientific | |
| Digitonin | G9441 | Promega | |
| MgCl2 | AM9530G | Thermo Fisher Scientific | |
| NP40 | 11332473001 | Sigma-Aldrich | |
| EDTA Solution pH 8.0 | AB00502 | AmericanBio | |
| Bovine Serum Albumin (BSA) | A8806 | Sigma-Aldrich | |
| Triton X-100 | T8787 | Sigma-Aldrich | |
| T4 DNA Ligase | M0202L | New England Biolabs | |
| T4 DNA Ligase Reaction Buffer | B0202S | New England Biolabs | |
| NEBuffer 3.1 | B7203S | New England Biolabs | |
| DPBS | 14190144 | Thermo Fisher Scientific | |
| Proteinase K | EO0491 | Thermo Fisher Scientific | |
| Ampure XP beads | A63880 | Beckman Coulter | |
| NEBNext High-Fidelity 2X PCR Master Mix | M0541L | New England Biolabs | |
| SYBR Green I Nucleic Acid Gel Stain | S7563 | Thermo Fisher Scientific | |
| DNA Clean & Concentrator-5 | D4014 | Zymo Research | |
| Tn5 Transposase - unloaded | C01070010 | Diagenode | |
| Tagmentation Buffer (2x) | C01019043 | Diagenode | |
| Sodium dodecyl sulfate | 71736 | Sigma-Aldrich | |
| Maxima H Minus Reverse Transcriptase (200 U/L) | EP0751 | Thermo Fisher Scientific | |
| dNTP mix | R0192 | Thermo Fisher Scientific | |
| SUPERased In RNase Inhibitor | AM2694 | Thermo Fisher Scientific | |
| Ampure XP beads | A63880 | Beckman Coulter | |
| Dynabeads MyOne C1 | 65001 | Thermo Fisher Scientific | |
| RNase Inhibitor | Y9240L | Enzymatics | |
| Kapa Hotstart HiFi ReadyMix | KK2601 | Kapa Biosystems | |
| Nextera XT DNA Preparation Kit | FC-131-1024 | Illumina |
Table S1A. List of reagents
| Oligo Name | Sequence | |
| Ligation Linker | CGAATGCTCTGGCCTCTCAAGCACGTGGAT | |
| P5 oligo | AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | |
| P7 oligo (701) | CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| P7 oligo (702) | CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| P7 oligo (703) | CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| P7 oligo (704) | CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| P7 oligo (705) | CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| P7 oligo (706) | CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT |
Table S2A. List of PCR Oligo.
| A | B | |
| Barcode A-1 | /5Phos/AGGCCAGAGCATTCGAACGTGATTTTTTTTTTTTTTTTVN | |
| Barcode A-2 | /5Phos/AGGCCAGAGCATTCGAAACATCGTTTTTTTTTTTTTTTVN | |
| Barcode A-3 | /5Phos/AGGCCAGAGCATTCGATGCCTAATTTTTTTTTTTTTTTVN | |
| Barcode A-4 | /5Phos/AGGCCAGAGCATTCGAGTGGTCATTTTTTTTTTTTTTTVN | |
| Barcode A-5 | /5Phos/AGGCCAGAGCATTCGACCACTGTTTTTTTTTTTTTTTTVN | |
| Barcode A-6 | /5Phos/AGGCCAGAGCATTCGACATTGGCTTTTTTTTTTTTTTTVN | |
| Barcode A-7 | /5Phos/AGGCCAGAGCATTCGCAGATCTGTTTTTTTTTTTTTTTVN | |
| Barcode A-8 | /5Phos/AGGCCAGAGCATTCGCATCAAGTTTTTTTTTTTTTTTTVN | |
| Barcode A-9 | /5Phos/AGGCCAGAGCATTCGCGCTGATCTTTTTTTTTTTTTTTVN | |
| Barcode A-10 | /5Phos/AGGCCAGAGCATTCGACAAGCTATTTTTTTTTTTTTTTVN | |
| Barcode A-11 | /5Phos/AGGCCAGAGCATTCGCTGTAGCCTTTTTTTTTTTTTTTVN | |
| Barcode A-12 | /5Phos/AGGCCAGAGCATTCGAGTACAAGTTTTTTTTTTTTTTTVN | |
| Barcode A-13 | /5Phos/AGGCCAGAGCATTCGAACAACCATTTTTTTTTTTTTTTVN | |
| Barcode A-14 | /5Phos/AGGCCAGAGCATTCGAACCGAGATTTTTTTTTTTTTTTVN | |
| Barcode A-15 | /5Phos/AGGCCAGAGCATTCGAACGCTTATTTTTTTTTTTTTTTVN | |
| Barcode A-16 | /5Phos/AGGCCAGAGCATTCGAAGACGGATTTTTTTTTTTTTTTVN | |
| Barcode A-17 | /5Phos/AGGCCAGAGCATTCGAAGGTACATTTTTTTTTTTTTTTVN | |
| Barcode A-18 | /5Phos/AGGCCAGAGCATTCGACACAGAATTTTTTTTTTTTTTTVN | |
| Barcode A-19 | /5Phos/AGGCCAGAGCATTCGACAGCAGATTTTTTTTTTTTTTTVN | |
| Barcode A-20 | /5Phos/AGGCCAGAGCATTCGACCTCCAATTTTTTTTTTTTTTTVN | |
| Barcode A-21 | /5Phos/AGGCCAGAGCATTCGACGCTCGATTTTTTTTTTTTTTTVN | |
| Barcode A-22 | /5Phos/AGGCCAGAGCATTCGACGTATCATTTTTTTTTTTTTTTVN | |
| Barcode A-23 | /5Phos/AGGCCAGAGCATTCGACTATGCATTTTTTTTTTTTTTTVN | |
| Barcode A-24 | /5Phos/AGGCCAGAGCATTCGAGAGTCAATTTTTTTTTTTTTTTVN | |
| Barcode A-25 | /5Phos/AGGCCAGAGCATTCGAGATCGCATTTTTTTTTTTTTTTVN | |
| Barcode A-26 | /5Phos/AGGCCAGAGCATTCGAGCAGGAATTTTTTTTTTTTTTTVN | |
| Barcode A-27 | /5Phos/AGGCCAGAGCATTCGAGTCACTATTTTTTTTTTTTTTTVN | |
| Barcode A-28 | /5Phos/AGGCCAGAGCATTCGATCCTGTATTTTTTTTTTTTTTTVN | |
| Barcode A-29 | /5Phos/AGGCCAGAGCATTCGATTGAGGATTTTTTTTTTTTTTTVN | |
| Barcode A-30 | /5Phos/AGGCCAGAGCATTCGCAACCACATTTTTTTTTTTTTTTVN | |
| Barcode A-31 | /5Phos/AGGCCAGAGCATTCGGACTAGTATTTTTTTTTTTTTTTVN | |
| Barcode A-32 | /5Phos/AGGCCAGAGCATTCGCAATGGAATTTTTTTTTTTTTTTVN | |
| Barcode A-33 | /5Phos/AGGCCAGAGCATTCGCACTTCGATTTTTTTTTTTTTTTVN | |
| Barcode A-34 | /5Phos/AGGCCAGAGCATTCGCAGCGTTATTTTTTTTTTTTTTTVN | |
| Barcode A-35 | /5Phos/AGGCCAGAGCATTCGCATACCAATTTTTTTTTTTTTTTVN | |
| Barcode A-36 | /5Phos/AGGCCAGAGCATTCGCCAGTTCATTTTTTTTTTTTTTTVN | |
| Barcode A-37 | /5Phos/AGGCCAGAGCATTCGCCGAAGTATTTTTTTTTTTTTTTVN | |
| Barcode A-38 | /5Phos/AGGCCAGAGCATTCGCCGTGAGATTTTTTTTTTTTTTTVN | |
| Barcode A-39 | /5Phos/AGGCCAGAGCATTCGCCTCCTGATTTTTTTTTTTTTTTVN | |
| Barcode A-40 | /5Phos/AGGCCAGAGCATTCGCGAACTTATTTTTTTTTTTTTTTVN | |
| Barcode A-41 | /5Phos/AGGCCAGAGCATTCGCGACTGGATTTTTTTTTTTTTTTVN | |
| Barcode A-42 | /5Phos/AGGCCAGAGCATTCGCGCATACATTTTTTTTTTTTTTTVN | |
| Barcode A-43 | /5Phos/AGGCCAGAGCATTCGCTCAATGATTTTTTTTTTTTTTTVN | |
| Barcode A-44 | /5Phos/AGGCCAGAGCATTCGCTGAGCCATTTTTTTTTTTTTTTVN | |
| Barcode A-45 | /5Phos/AGGCCAGAGCATTCGCTGGCATATTTTTTTTTTTTTTTVN | |
| Barcode A-46 | /5Phos/AGGCCAGAGCATTCGGAATCTGATTTTTTTTTTTTTTTVN | |
| Barcode A-47 | /5Phos/AGGCCAGAGCATTCGCAAGACTATTTTTTTTTTTTTTTVN | |
| Barcode A-48 | /5Phos/AGGCCAGAGCATTCGGAGCTGAATTTTTTTTTTTTTTTVN | |
| Barcode A-49 | /5Phos/AGGCCAGAGCATTCGGATAGACATTTTTTTTTTTTTTTVN | |
| Barcode A-50 | /5Phos/AGGCCAGAGCATTCGGCCACATATTTTTTTTTTTTTTTVN |
Table S2B. List of DNA Barcode A Sequences.
| A | B | |
| Barcode B-1 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACGTGATATCCACGTGCTTGAG | |
| Barcode B-2 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAACATCGATCCACGTGCTTGAG | |
| Barcode B-3 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATGCCTAAATCCACGTGCTTGAG | |
| Barcode B-4 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTGGTCAATCCACGTGCTTGAG | |
| Barcode B-5 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACCACTGTATCCACGTGCTTGAG | |
| Barcode B-6 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACATTGGCATCCACGTGCTTGAG | |
| Barcode B-7 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAGATCTGATCCACGTGCTTGAG | |
| Barcode B-8 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCATCAAGTATCCACGTGCTTGAG | |
| Barcode B-9 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGCTGATCATCCACGTGCTTGAG | |
| Barcode B-10 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACAAGCTAATCCACGTGCTTGAG | |
| Barcode B-11 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGTAGCCATCCACGTGCTTGAG | |
| Barcode B-12 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTACAAGATCCACGTGCTTGAG | |
| Barcode B-13 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACAACCAATCCACGTGCTTGAG | |
| Barcode B-14 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACCGAGAATCCACGTGCTTGAG | |
| Barcode B-15 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACGCTTAATCCACGTGCTTGAG | |
| Barcode B-16 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAGACGGAATCCACGTGCTTGAG | |
| Barcode B-17 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAGGTACAATCCACGTGCTTGAG | |
| Barcode B-18 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACACAGAAATCCACGTGCTTGAG | |
| Barcode B-19 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACAGCAGAATCCACGTGCTTGAG | |
| Barcode B-20 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACCTCCAAATCCACGTGCTTGAG | |
| Barcode B-21 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACGCTCGAATCCACGTGCTTGAG | |
| Barcode B-22 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACGTATCAATCCACGTGCTTGAG | |
| Barcode B-23 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACTATGCAATCCACGTGCTTGAG | |
| Barcode B-24 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGAGTCAAATCCACGTGCTTGAG | |
| Barcode B-25 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGATCGCAATCCACGTGCTTGAG | |
| Barcode B-26 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGCAGGAAATCCACGTGCTTGAG | |
| Barcode B-27 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTCACTAATCCACGTGCTTGAG | |
| Barcode B-28 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATCCTGTAATCCACGTGCTTGAG | |
| Barcode B-29 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATTGAGGAATCCACGTGCTTGAG | |
| Barcode B-30 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAACCACAATCCACGTGCTTGAG | |
| Barcode B-31 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGACTAGTAATCCACGTGCTTGAG | |
| Barcode B-32 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAATGGAAATCCACGTGCTTGAG | |
| Barcode B-33 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCACTTCGAATCCACGTGCTTGAG | |
| Barcode B-34 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAGCGTTAATCCACGTGCTTGAG | |
| Barcode B-35 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCATACCAAATCCACGTGCTTGAG | |
| Barcode B-36 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCAGTTCAATCCACGTGCTTGAG | |
| Barcode B-37 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCGAAGTAATCCACGTGCTTGAG | |
| Barcode B-38 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCGTGAGAATCCACGTGCTTGAG | |
| Barcode B-39 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCTCCTGAATCCACGTGCTTGAG | |
| Barcode B-40 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGAACTTAATCCACGTGCTTGAG | |
| Barcode B-41 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGACTGGAATCCACGTGCTTGAG | |
| Barcode B-42 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGCATACAATCCACGTGCTTGAG | |
| Barcode B-43 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTCAATGAATCCACGTGCTTGAG | |
| Barcode B-44 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGAGCCAATCCACGTGCTTGAG | |
| Barcode B-45 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGGCATAATCCACGTGCTTGAG | |
| Barcode B-46 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGAATCTGAATCCACGTGCTTGAG | |
| Barcode B-47 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAAGACTAATCCACGTGCTTGAG | |
| Barcode B-48 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGAGCTGAAATCCACGTGCTTGAG | |
| Barcode B-49 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGATAGACAATCCACGTGCTTGAG | |
| Barcode B-50 | /5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGCCACATAATCCACGTGCTTGAG |
Table S2C. List of DNA Barcode B Sequences.
- This protocol requires using a microfluidic device fabricated with PDMS using soft lithography.
- A hole punching machine (SCHMIDT® Manual Press) is needed to punch the inlet and outlet holes in the PDMS chip after its fabrication.
- The tissue of interest should be placed on the center of a poly-L-lysine coated glass slide. (CatLog no. 63478-AS, electron microscopy sciences).
- A custom-designed acrylic clamp with screws is needed to hold the PDMS device and the glass slide together firmly.
- The silicon wafer used for fabricating the PDMS mold is purchased from WaferPro (CatLog. No. C04004).
- The photoresist is purchased from MicroChem Laboratory (CatLog. No. SU-8 2010).
- A homemade laboratory vacuum system(See Figure Below) is needed to applying suction to move fluid in the microfluidic channels.
- Microscopy – The tissue of interest can be scanned and imaged using EVOS (Thermo Fisher EVOS fl), typically at a magnification of 10×. Any suitable optical microscope can be used.
- A Laser Engraver Cutter is used to cut out the acrylic barcode and lysis clamps.
- A “humidified chamber” (See Figure Below) to prevent reagent evaporation during incubation.
Key equipment used in DBiT-seq
Hole punch machine, the key device for DBiT-seq, and the house vacuum lines. (Left) SCHMIDT® Manual Press used for hole punching 2-mm diameter holes for the inlets and outlets of Chip-A and Chip-B. (Top Right) Example of a “humidified chamber” to prevent reagent evaporation during the incubation steps. (Bottom Right) House vacuum used for pulling reagents through the microchannels.
Recipes
Critical: Prepare the RT mix, template switch mix, ligation mix, PCR mix, 1X lysis solution, flow wash buffer, PBS-RI, 80% ethanol on the same day as usage. 2X lysis buffer, 1X B&W Buffer with 0.05% Tween-20, and 2X B&W Buffer can be stored for up to 6 months at room temperature.
PBS-RI
| Reagent | Volume (μL) | |
| 1X PBS | 5000 | |
| RNase Inhibitor (40 U/µL) (Enzymatics) | 7 | |
| Total | 5007 |
80% Ethanol
| Reagent | Volume(ml) | |
| RNase-free water | 1 | |
| 100% Ethanol | 4 | |
| Total | 5 |
RT Mixture
| Reagent | Volume (μL) | |
| 5X Maxima RT buffer | 50 | |
| RNase-free water | 32.8 | |
| RNase Inhibitor (Enzymatics) | 1.6 | |
| Superase In RNase Inhibitor (Ambion) | 3.2 | |
| dNTPs (10 mM stock) | 12.5 | |
| Maxima H Minus Reverse Transcriptase | 25 | |
| PBS-RI | 100 | |
| Total | 225.1 |
Ligation Mix
| Reagent | Volume (μL) | |
| 10X T4 Ligase Buffer | 27 | |
| 1X NEB buffer 3.1 with 1% RI (Enzymatics) | 115.8 | |
| 5% Triton-X100 | 5.4 | |
| RNase Inhibitor (Enzymatics) | 2.2 | |
| RNase-free water | 69.5 | |
| SuperaseIn RNase Inhibitor (Ambion) | 0.7 | |
| T4 DNA Ligase (400 U/µL) | 11 | |
| Total | 231.6 |
Template switch mix
| Reagent | Volume (μL) | |
| 20% Ficoll PM-400 | 44 | |
| 5X Maxima RT buffer | 44 | |
| dNTPs (10 mM stock) | 22 | |
| RNase Inhibitor (Enzymatics) | 5.5 | |
| Maxima H Minus Reverse Transcriptase | 11 | |
| Template Switch Primer (100 μM stock) | 5.5 | |
| RNase-free water | 88 | |
| Total | 220 |
PCR mix
| Reagent | Volume (μL) | |
| 2X Kapa Hifi HotStart master mix | 110 | |
| Primer1 BC_0062 (10μM) | 8.8 | |
| Primer2 BC_0108 (10μM) | 8.8 | |
| RNase-free water | 92.4 | |
| Total | 220 |
Flow Wash Buffer
| Reagent | Volume (mL) | |
| 1X PBS | 4 | |
| 10 % Triton X-100 | 0.04 | |
| Superase In RNase Inhibitor | 0.01 | |
| Total volume | 4.05 |
1X Lysis Solution
| Reagent | Volume (μL) | |
| 1X PBS | 50 | |
| 2X Lysis Buffer | 50 | |
| Proteinase K (20mg/mL) (Thermo) | 10 | |
| Total | 110 |
2X Lysis Buffer
| Reagent | Stock Concentration | Final Concentration (2X) | Volume (mL) | |
| Tris, pH 8.0 | 1 M | 20 mM | 0.5 | |
| NaCl | 5 M | 400 mM | 2 | |
| EDTA, pH 8.0 | 0.5 M | 100 mM | 5 | |
| SDS | 10% | 4.4 % | 11 | |
| RNase-free Water | NA | NA | 6.5 | |
| Final Volume | 25 |
1X B&W Buffer with 0.05% Tween-20
| Reagent | Volume | |
| 1M Tris-HCl pH 8.0 | 100 μL | |
| EDTA, 0.5M | 20 μL | |
| 5M NaCl | 4 mL | |
| Tween 20 10% | 100 μL | |
| RNase-free water | 15.78 mL | |
| Total | 20 mL |
2X B&W Buffer
| Reagent | Volume | |
| 1M Tris-HCl pH 8.0 | 500 μL | |
| EDTA, 0.5M | 100 μL | |
| 5M NaCl | 20 mL | |
| RNase-free water | 29.4 mL | |
| Total | 50 mL |
CRITICAL: Handle all in an RNase-free area.
Troubleshooting
Before start
The protocol below describes the reagents, equipment, and specific experimental steps for using the spatial-ATAC-seq and RNA-seq platform on fixed fresh frozen tissue slides. The 20 μm microfluidic channel width were designed and validated to provide spatial epigenomic and transcriptome 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.
Fabricating the Silicon Wafer Device Mold
12h
Prepare chrome photomasks of the microfluidics chip by printing the high-resolution CAD files onto a glass substrate (Figure 1).
Figure 1: Microfluidic Photomask Design.
(Left) AutoCAD design for the 20 micron-width fifty channel Chip-A and Chip-B. (Right) AutoCAD design for 10 micron-width channel Chip-A and Chip-B.
NOTE: We have outsourced this step (Front Range Photomask, USA).
Using these masks, prepare a replica mold as follows:
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.
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)
Bake the wafer at 180°C for 10 min on a hot plate to dry it out.
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.
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.
Expose the SU-8 on the wafer through the photomask using Mask Aligner with a dose of 150 mJ/cm2 UV.
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.
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).
Rinse the wafer with 100% isopropanol and dry with compressed air.
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.
Creating the Acrylic Clamps
30m
Prepare the pattern and dimensions for the barcoding clamp and lysis clamp.
Peel covering of acrylic sheet and place it in the laser cutting machine.
Select the program dimensions and cut two pieces for the top and bottom of the barcoding clamp.
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.
Remove cut-out scraps with a pipet tip or a similar pointed tool.
Repeat steps 15 and 16 for the bottom blank piece.
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).
Figure 2. Key parts for setup. PDMS reservoirs and acrylic clamps used in DBiT-seq. (A) Inlet Reservoir. (B) Barcoding Clamp. (C) Lysis Reservoir. (D) Lysis Clamp.
NOTE: The acrylic clamps can be reused indefinitely.
Preparing the Microfluidic Device and reagent reservoirs
3h
Thoroughly mix polydimethylsiloxane (PDMS) elastomer base and curing agent (these come together) at a 10:1 ratio (See Figure 3).
Figure 3: Step-by-step visual guide for making Chip-A and Chip-B.
Pour the mixture into the silicon device mold.
NOTE: Aim for a chip height of about 5mm.
Place in a vacuum desiccator until all bubbles dissipate from the mixture about 30-60 min.
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!
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.
Thoroughly clean the surface of the device with scotch tape.
Pour mixture in a container large enough to cut out two roughly 25x25x5mm PDMS pieces.
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.
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.
Preparing Barcodes (A and B).
1h
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.
Place the 50 mixes in a thermal cycle and heat to 97˚C to anneal.
Slowly cool to room temperature at a rate of -0.1˚C/sec.
Store at -20˚C for up to 6 months.
Preparing the Tissue Slide
1d
25% sucrose overnight bath.
Embed in OCT frozen on dry ice.
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.
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.
Transposome assembly preparation
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.
Resuspend the oligos ( Tn5Erev, Tn5ME-A , and Tn5ME-B) in Annealing Buffer to stock
concentration of 100 µM.
In a PCR tube, mix 10 μl of oligo Rev with 10 μl of oligo A.
In a separate PCR tube, mix 10 μl of oligo Rev with 10 μl of oligo B
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.
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.
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.
Tissue Preparation
1h
Remove stored sections from the freezer and allow to warm to room temperature for 10 minutes.
Clean sections by pipetting 2 mL of PBS-RI across the tissue.
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.
Remove the formaldehyde by pipetting it off and quench the tissue with 1mL 1.25M glycine for 5
min at room temperature.
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.
Dry the section with gentle air flow.
Take a full pre-scan image of the section at the desired optical resolution (Figure 4A).
Recommended 10X resolution.
Figure 4 : Example scanning of the tissue slide.
(A) Initial full scan of the tissue section prior to DBiT-seq.
Attach the Antibody PDMS reservoir and 12mm diameter clamp to the tissue.
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)
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)
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
| A | B | C | D | |
| 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) |
Incubate the tissue at 37 ºC for 30 min
Stop the tagmentation reaction by adding 500 μL of 40 mM EDTA onto the tissue and incubating at room temperature for 5 min.
Dip in a 50 mL falcon tube with DI water and gently dry with air flow.
Bulk Tissue Reverse Transcription
Primer sequences: 15T Biotin RT Primer:
/5Phos/CATCGGCGTACGACTNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTTVN
Reverse transcription mix as below
Reverse transcription mix
The 60μL RT Mix was loaded on the sample with PDMS reservoir and sealed with parafilm. Then, incubated at room temperature for 30 min, and 42˚C for 90 min, in a wet box.
1X NEB buffer 3.1: 100μL 10X NEB buffer + 900μL Nuclease-free Water.
After RT reaction, samples were flow washed with 1 mL Nuclease-free Water for 5 times, then incubated in 500μL 1X NEB buffer 3.1 for 5 min at room temperature, followed by air drying.
DBiT-seq assembly and Barcoding
Lysis
Take a small PDMS solution reservoir and place it on the tissue slide so that the reservoir opening is directly over the barcoded ROI.
Load the slide into a lysis clamping device and tighten screws as much as possible by hand.
Image the lysis chip on the ROI using a 10x objective lens.
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 | - | - |
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.
Cover the ROI hole using parafilm and load the slide into a humidifying chamber.
Incubate tissue the slide with PDMS reservoir and Lysis Solution at 58 ºC for 2 hours to reverse formaldehyde crosslinks.
After incubation, collect the lysate in a 200μL PCR tube (or 1.5mL tube) and seal the tube with parafilm.
Incubate the lysate at 65 ºC with rotation overnight.
Purification
If necessary, transfer the lysate to a 1.5mL micro-centrifuge tube.
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.
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
cDNA Purification
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.
Wash 3 times with 800 µL of 1X B&W buffer with 0.05% Tween-20.
Resuspend beads in 100 µL 2X B&W buffer + 2 µL SUPERase• InTM RNase Inhibitor.
Purify lysate following the DNA Clean & Concentrator with Zymo-spin IC Columns (RPI Research Products) protocol. Use 100 µL water to elute DNA.
Add 100 µL of resuspended DynabeadsTM MyOneTM Streptavidin C1 magnetic beads to each lysate.
Rotate at 30 RPM and room temperature for 60 min for binding to occur.
Save the supernatant for the spatial-ATAC-seq library
Wash twice with 400 µL of 1X B&W buffer with 5 min rotation after resuspending beads.
Wash once more with 400 µL 10 mM Tris containing 0.1% Tween-20 with 5 min rotation after resuspension.
Template Switch
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.
Rotate beads at 30 RPM and room temperature for 30 min followed by rotation at 42˚C for 90 min.
PCR fro
Wash beads and resuspend.
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.
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.
Perform PCR to detach cDNA from beads.
PCR Cycling Conditions
Remove the DynabeadsTM from the PCR solution using a magnetic tube holder.
Add Evagreen (Biotium) at a 1X concentration.
NOTE: This step is optional.
Perform PCR again with the following thermocycling conditions. Cycling can also be halted once the qPCR signal begins to plateau.
cDNA Purification
Add 80 µL KAPA pure beads for 100 µL of the cDNA sample.
Mix thoroughly by pipetting or vortexing.
Incubate the tubes at room temperature for at least 10 min to bind the cDNA to the beads.
Keep the tubes on the magnet and add 200 µL of 80% ethanol.
Carefully remove and discard the supernatant.
Incubate the tubes at room temperature for at least 30 sec.
Carefully remove and discard the ethanol.
Repeat steps 102-104.
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.
Incubate the tubes at room temperature for at least 10 min to elute the cDNA off the beads.
Place the tubes back on the magnet and incubate until the liquid is fully clear.
Transfer the clear supernatant to a new tube.
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.
Library Preparation
PCR product from previous step can be sequenced.
Purification of spatial-ATAC-seq product
If necessary, transfer the lysate to a 1.5mL microcentrifuge tube.
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.
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 21 uL of DNA Elution buffer
PCR for spatial-ATAC-seq
2h
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
| Steps | Temperature | Time | Cycles | |
| Initial Denaturation | 72˚C | 5 min | 1 | |
| Denaturation | 98˚C | 30 sec | ||
| Denaturation | 98˚C | 10 sec | 5 | |
| Annealing | 63˚C | 30 sec | ||
| Extension | 72˚C | 1 min |
PCR Cycling Conditions
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.
Repeat step 68 PCR protocol to amplify the remaining product with the cycles determined in step 69
Ampure Purification and Library preperation for spatial-ATAC-seq
40m
Perform a 1X SPRI purification by adding 45uL Ampure XP beads to the sample. Mix thoroughly by pipetting or vortexing.
Mix thoroughly by pipetting.
Keep the tubes on the magnet and add 200 µL of 80% ethanol.
Carefully remove and discard the supernatant.
Incubate the tubes at room temperature for at least 30 sec.
Carefully remove and discard the ethanol.
Repeat steps 102-104102-.
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.
Incubate the tubes at room temperature for at least 10 min to elute the cDNA off the beads.
Place the tubes back on the magnet and incubate until the liquid is fully clear.
Transfer the clear supernatant to a new tube.
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.