Jan 07, 2025
Spatial Multi-omics Sequencing for Formalin-Fixed Paraffin Embedded Tissue via DBiT-seq
- Archibald Enninful1,
- Rong Fan1,
- Negin Farzad1
- 1Yale University
- Fan Lab Yale
External link: https://www.cell.com/cell/fulltext/S0092-8674(24)01019-5
Protocol Citation: Archibald Enninful, Rong Fan, Negin Farzad 2025. Spatial Multi-omics Sequencing for Formalin-Fixed Paraffin Embedded Tissue via DBiT-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger2wwl47/v1
Manuscript citation:
Bai et al., 2024, Cell 187, 6760–6779
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 06, 2025
Last Modified: January 07, 2025
Protocol Integer ID: 117754
Funders Acknowledgements:
SenNet Consortium
Grant ID: 1U54AG076043-01
Abstract
This protocol describes the use of Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq) platform to construct a multi-omics atlas on FFPE tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics.
This protocol is adapted from the Patho-DBiT publication - Spatially exploring RNA biology in archival formalin fixed paraffin-embedded tissues (Bai et al., 2024, Cell 187, 6760–6779)
Image Attribution
Image from graphical abstract of this publication - Bai et al., 2024, Cell 187, 6760–6779
Materials
KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| TotalSeqTM antibodies | Biolegend | See Table 1 | |
| Chemicals, Peptides, and Recombinant Proteins | |||
| Maxima H Minus Reverse Transcriptase (200 U/L) | Thermo Fisher Scientific | EP0751 | |
| dNTP mix | Thermo Fisher Scientific | R0192 | |
| RNase Inhibitor | Enzymatics | Y9240L | |
| SUPERase• InTM RNase Inhibitor | Thermo Fisher Scientific | AM2694 | |
| T4 DNA Ligase | New England Biolabs | M0202L | |
| KAPA Pure Beads | Kapa Biosystems | KK8002 | |
| DynabeadsTM MyOneTM Streptavidin C1 | Thermo Fisher Scientific | 65001 | |
| Proteinase K, recombinant, PCR grade | Thermo Fisher Scientific | EO0491 | |
| Kapa HiFi Hotstart ReadyMix PCR Kit | Kapa Biosystems | KK2601 | |
| Formaldehyde solution | Sigma | F8775-25ML | |
| NEBuffer 3.1 | New England Biolabs | B7203S | |
| T4 DNA Ligase Reaction Buffer | New England Biolabs | B0202S | |
| Evagreen Dye, 20X in water | Biotium | 31000-T | |
| Oligonucleotides | |||
| Primers, Ligation linkers, DNA barcodes | IDT | See Table 2 | |
| Critical Commercial Assays | |||
| DNA Clean & Concentrator with Zymo-Spin I Columns | Research Products International | ZD4004 | |
| Nextera XT DNA Preparation Kit | Illumina | FC-131-1024 | |
| Other | |||
| Multiplate PCR Plate, 96-well, clear | BioRad | MLL9601 | |
| 50 mL Falcon Tube | Corning | 352070 | |
| BioDot Pure 8 – Strip PCR tubes w/ Optically Clear Flat Caps | Dot Scientific Inc. | 403-8PCR | |
| SYLGARDTM 184 Silicone Elastomer Base and Curing Agent | Dow Corning | 4019862 | |
| SuperChipTM Poly-L-Lysine Slides | Electron Microscopy Sciences | 63478-AS | |
| RNaseZapTM RNase Decontamination Solution | Thermo Fisher Scientific | AM9780 | |
| Silicon wafer | WaferPro | C04004 | |
| Photoresist SU-8 2010 | MicroChem Laboratory | SU-8 2010 |
| Category | Barcode | Specificity | Clone | Barcode Sequence | |
| TotalSeqTM-A | 12 | CD117 (c-kit) | 2B8 | TGCATGTCATCGGTG | |
| TotalSeqTM-A | 78 | CD49d | R1-2 | CGCTTGGACGCTTAA | |
| TotalSeqTM-A | 96 | CD45 | 30-F11 | TGGCTATGGAGCAGA | |
| TotalSeqTM-A | 104 | CD102 | 3C4 (MIC2/4) | GATATTCAGTGCGAC | |
| TotalSeqTM-A | 115 | FcεRIα | MAR-1 | AGTCACCTCGAAGCT | |
| TotalSeqTM-A | 118 | NK-1.1 | avas12 | GTAACATTACTCGTC | |
| TotalSeqTM-A | 119 | Siglec H | 551 | CCGCACCTACATTAG | |
| TotalSeqTM-A | 122 | TER-119/Erythroid Cells | TER-119 | GCGCGTTTGTGCTAT | |
| TotalSeqTM-A | 130 | Ly-6A/E (Sca-1) | D7 | TTCCTTTCCTACGCA | |
| TotalSeqTM-A | 232 | MAdCAM-1 | MECA-367 | TTGGGCGATTAAGAA | |
| TotalSeqTM-A | 381 | Panendothelial Cell Antigen | MECA-32 | CGTCCTAGTCATTGG | |
| TotalSeqTM-A | 415 | P2RY12 | S16007D | TTGCTTATTTCCGCA | |
| TotalSeqTM-A | 439 | CD201 (EPCR) | RCR-16 | TATGATCTGCCCTTG | |
| TotalSeqTM-A | 442 | Notch 1 | HMN1-12 | TCCGGTCACTCAGTA | |
| TotalSeqTM-A | 443 | CD41 | MWReg30 | ACTTGGATGGACACT | |
| TotalSeqTM-A | 449 | CD326 (Ep-CAM) | G8.8 | ACCCGCGTTAGTATG | |
| TotalSeqTM-A | 552 | CD304 (Neuropilin-1) | 3E12 | CCAGCTCATTCAACG | |
| TotalSeqTM-A | 553 | CD309 (VEGFR2, Flk-1) | Avas12 | ATAAGAGCCCACCAT | |
| TotalSeqTM-A | 558 | CD55 (DAF) | RIKO-3 | ATTGTTGTCAGACCA | |
| TotalSeqTM-A | 559 | CD63 | NVG-2 | ATCCGACACGTATTA | |
| TotalSeqTM-A | 564 | Folate Receptor β (FR-β) | 10/FR2 | CTCAGATGCCCTTTA | |
| TotalSeqTM-A | 596 | ESAM | 1G8/ESAM | TATAGTTTCCGCCGT |
Table S1. TotalSeqTMAntibodies, related to Step 9.
| Oligo Name | Sequence | |
| Primer 1 | CAAGCGTTGGCTTCTCGCATCT | |
| Primer 2 | AAGCAGTGGTATCAACGCAGAGT | |
| Primer 3 (cite-seq) | CCTTGGCACCCGAGAATT*C*C | |
| Ligation Linker | CGAATGCTCTGGCCTCTCAAGCACGTGGAT | |
| Template Switch Primer | AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G | |
| P5 oligo | AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | |
| P5 oligo (cite-seq) | AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTTGGCACCCGAGAATTCCA | |
| 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.
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.
Fabricating the Silicon Wafer Device Mold
Fabricating the Silicon Wafer Device Mold
12h
12h
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)
50Prepare 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 25 micron-width 50 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.
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
Creating the Acrylic Clamps
30m
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
Preparing the Microfluidic Device and reagent reservoirs
3h
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
Preparing Barcodes
1h
1h
Thoroughly mix the ligation linker with each barcodes A1-50 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
Preparing the Tissue Slide
1d
1d
FFPE tissue sections were cut into sections with a thickness < 10 um and were placed at the center of a poly-L-lysine glass slide. Sections were stored at -80 ˚C until use.
Tissue sections were taken out from the freezer and allowed to come up to room temperature for 10 min until all the moisture disappears.
Tissue Preparation
Tissue Preparation
1h
1h
Bake slides at 60 degrees Celsius for 2 hours.
Perform deparaffinization as follows:
* 100% Xylene 5 minutes
* 100% Xylene 5 minutes
* 100% Ethanol for 5 minutes
* 90% Ethanol for 5 minutes
* 70% Ethanol for 5 minutes
* 50% Ethanol for 5 minutes
* Deionized water for 5 minutes
5. Antigen-retrieval using 1X Tris-EDTA pH 9
* Allow to boil
* Incubate slide for
~ 10 minutes
* Allow to cool to room temperature (Can incorporate cooling on ice to speed up the process)
Permeabilization: 500μL 0.5% Triton X-100 in PBS was added on top of the tissue, and tissues were permeabilized for 20 min at room temperature.
To stop permeabilization, 0.5X PBS-RI was used to clean up the tissue sections.
Take a full pre-scan image of the section at the desired optical resolution
Recommended 10X resolution.
In situ polyadenylation
In situ polyadenylation
Reagents
Poly(A) wash buffer: 20μL 5X Poly(A) Polymerase Reaction Buffer, 2μL RNase Inhibitor, 78μl nuclease-free water.
| A | B | C | D | |
| Reagent name | Concentration | For 1 chip | For 2 chips | |
| Nuclease-free Water | 47.6μL | 95.3μL | ||
| 5X Poly(A) Reaction Buffer | 5X | 15μL | 30μL | |
| Poly(A) Polymerase | 600U/μL | 3μL | 6μL | |
| ATP | 100mM | 0.38μL | 0.75μL | |
| SuperaseIn RNase Inhibitor | 20U/μL | 6μL | 12μL | |
| RNase Inhibitor | 40U/μL | 3μL | 6μL |
Table 1: Poly(A) mix preparation
The tissue section was equilibrated by adding 100μL of Poly(A) wash buffer, incubated for 5 min at room temperature, followed by air drying.
Next, attach the PDMS reservoir, add 75μL of Poly(A) Mix to the reaction chamber, and seal with
parafilm. Incubate at 37˚C for 25 min, in a wet box.
After incubation, 0.5X PBS-RI was used to clean up the tissue sections.
Bulk Tissue Reverse Transcription
Bulk Tissue Reverse Transcription
1h
1h
Dip in a 50 mL falcon tube with DI water and gently dry with air flow.
Primer sequences: 15T Biotin RT Primer:
/5Phos/CATCGGCGTACGACTNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTTVN
Reverse transcription mix
| A | B | C | D | |
| Reagent name | Concentration | For 1 chip | For 2 chips | |
| 0.5X PBS-RI | 20μL | 40μL | ||
| 5X Maxima RT buffer | 5X | 12μL | 24μL | |
| 30T Biotin RT Primer | 25uM | 10μL | 20μL | |
| Maxima H Minus RT | 200U/μL | 6μL | 12μL | |
| Nuclease-free Water | 8μL | 16μL | ||
| dNTPs (10mM) | 10mM | 3μL | 6μL | |
| SuperaseIn RNase Inhibitor | 20U/μL | 0.8μL | 1.6μL | |
| RNase Inhibitor | 40U/μL | 0.4μL | 0.8μL |
Table 2: Reverse Transcription (RT) mix preparation
1X NEB buffer 3.1: 100μL 10X NEB buffer + 900μL Nuclease-free Water.
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.
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.
Ligation of Barcodes
Ligation of Barcodes
Reagents
Ligation mix
| A | B | C | D | |
| Reagent name | Concentration | For 1 chip | For 2 chips | |
| 1X NEB buffer 3.1 | 1X | 100μL | 200μL | |
| 10X T4 Ligase buffer | 10X | 26μL | 52μL | |
| Nuclease-free Water | 61.3μL | 122.6μL | ||
| T4 DNA Ligase | 400U/μL | 15μL | 30μL | |
| 5% Triton-X100 | 5μL | 10μL | ||
| RNase Inhibitor | 40U/μL | 2μL | 4μL | |
| SuperaseIn RNase Inhibitor | 20U/μL | 0.7μL | 1.4μL |
Table 3: Preparation of ligation mix
Then, in a 96-well plate, add 4μL of the above Ligation Mix for each well.
For each well, 1μL of 25uM of prepared Barcode A with Linker1 (A1-A50) was added and mixed with the Ligation Mix.
Prepare the wash buffer as follows:
Wash buffer: 2mL PBS+20μL 10% Triton X-100+5μL SuperaseIn RNase Inhibitor.
Attaching PDMS chips and loading of ligation mix
Attaching PDMS chips and loading of ligation mix
1h
1h
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).
Figure 4. Assembly to create the setup for the PDMS reservoir and acrylic clamps. (A) Chip-A with the inlet reservoir and barcoding clamp. (B) Sideview of Chip-A. (C) The lysis reservoir with the lysis clamp on top of the barcoded tissue. (D) Sideview of the lysis setup.
Figure 5. Example Scanning of the Tissue Slide.
(C) Scan of Chip-A channels placed on the tissue.
Use the acrylic clamp to firmly hold the PDMS chip against the tissue.
Place the inlet reservoir above the inlets. The final mixture (5μL in total) was injected into each of the 50 inlets of the 1st PDMS and vacuumed through the channels, making sure all the channels were filled.
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.
Figure 6. Chip-A with Acrylic Clamp and House Vacuum attached.
Stop the vacuum and incubate at 37 ˚C for 15 mins. Take out the chips and check if there are
missed channels induced by bubbles. Vacuum again if needed and further incubate
for 30 mins.
After ligation reaction, aspirate remaining barcode A mixture from inlets/outlets.
Clean the channels by flushing 1mL Wash buffer for 5 min and peel off PDMS and flow wash the chip once with 1 mL Nuclease-free Water, followed
by air dry.
In-Tissue Ligation of barcode B
In-Tissue Ligation of barcode B
1h
1h
Attach Chip-B centered on the tissue.
Scan the channels over the tissue to ensure proper placement.
Figure 7: Example scanning of the tissue slide.
(D) Scan of Chip-B channels across the tissue.
Firmly clamp as before.
Prepare the ligation mixture.
Prepare barcode B mixture.
In a 96-well PCR plate, add 4 µL of the ligation mixture in each of 50 wells.
Add 1 µL of each 25 µM Barcode B1-50 to the wells and mix thoroughly.
Introduce final barcode B mixture to the tissue.
Pipet each barcode B mixture directly into the inlets.
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.
Incubate in the humidified chamber at 37˚C for 30 min for in-cell ligation of barcode B (Figure 8).
Clean the channels with wash buffer for 10 min.
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.
Peel off Chip-B and dip the slide into a 50mL falcon tube with DI water and gently dry with air flow.
Take a final post-scan of the tissue section.
Figure 8. Example Scanning of the Tissue Slide.
(B) Final scan after barcoding and removing the device.
Lysis and Sub-library Generation
Lysis and Sub-library Generation
2h 30m
2h 30m
Place the lysis reservoir directly around the barcoded tissue region.
NOTE: Try to cover as little extra tissue as possible.
Tightly apply the lysis clamp.
Add lysis solution to lysis reservoir depending on channel dimensions (~20 µL for the 25 µm channels, ~10 µL for the 10 µm channels).
Lysis solution is made with 50 µL 1X PBS, 50 µL of 2X lysis buffer, and 10 µL of proteinase K solution (20mg/mL).
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.
Place in the humidified chamber and tightly wrap the chamber with parafilm and incubate at 55˚C for 2 hours.
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.
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.
cDNA Purification
cDNA Purification
1h 30m
1h 30m
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.
Wash beads.
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
Template Switch
2h
2h
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
PCR
2h
2h
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.
| Steps | Temperature | Time | Cycles | |
| Initial Denaturation | 95˚C | 3 min | 1 | |
| Denaturation | 98˚C | 20 sec | 5 | |
| Annealing | 65˚C | 45 sec | ||
| Extension | 72˚C | 3 min |
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.
| Steps | Temperature | Time | Cycles | |
| 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 | ||
Removal of ribosomal RNA
Removal of ribosomal RNA
rRNA removal using Bio-Rad kit. Link: SEQuoia RiboDepletion Kit (17006487)
After rRNA removal, another 10 cycle PCR using 200uL PCR reaction system (100uL 2X Kapa
HiFi) using direct ligation primer and Illumina N70X primer
cDNA Purification
cDNA Purification
40m
40m
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.
Mix thoroughly by pipetting.
Incubate the tubes at room temperature for at least 10 min to bind the cDNA to the beads.
Place the tubes on a magnet to capture the beads and incubate until the liquid is clear.
Carefully remove and discard the supernatant.
Keep the tubes on the magnet and add 200 µL of 80% ethanol.
Incubate the tubes at room temperature for at least 30 sec.
Carefully remove and discard the ethanol.
Repeat steps 86-88.
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.
Remove the tubes from the magnet and resuspend the beads in 15 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
Library Preparation
1h 30m
1h 30m
PCR product from previous step can be sequenced.