Bill of Materials (BOM)¶
Complete reagent and materials list, organized by workflow stage. Use the tabs to find what you need for each step.
How to use
The workflow is: Pre-treatment → RCA → Readout → Morphology. Order everything from the stages you will perform.
Reagents for preparing tissue sections before RCA.
Shared (FF & FFPE)¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Paraformaldehyde (16% aq.) | EMS | 15710 | Dilute to 4% fresh |
| Pepsin (porcine gastric mucosa) | Sigma-Aldrich | P6887 | See Pepsin Prep |
| HCl (conc.) | Any | -- | 12 M; dilute to 0.1 M for pepsin |
| Ethanol (mol. bio. grade) | Any | -- | 70%, 80%, 85%, 100% series |
| PBS (1X) | Any | -- | RNase-free |
| Tween-20 | Sigma-Aldrich | P1379 | 0.05% in PBS for wash buffer |
| Nuclease-Free Water | Any | -- | |
| PDMS Chambers | Homemade; Sylgard 184 (Dow) | -- | See PDMS Fabrication |
| Microscope Slides | Superfrost Plus | -- | |
| RNaseZap | Invitrogen | AM9780 | Decontaminate all surfaces |
FFPE Only¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Xylene (or substitute) | Any (histology grade) | -- | Deparaffinization |
| Methanol | Any | -- | For methanol--acetic acid 3:1 |
| Acetic Acid (glacial) | Any | -- | |
| Sodium Citrate Buffer | Any | -- | 0.01 M pH 6; 80°C RNA retrieval |
| NaBH₄ (Sodium Borohydride) | Sigma-Aldrich | 213462 | 1% in PBS; prepare fresh on ice |
| Coverslips | Any | -- | FFPE section mounting |
| EasyDip Staining Jars | Any | -- | Decontaminate with RNaseZap |
NaBH₄ Safety
Explosive if liquid enters stock bottle. Prepare fresh each time on ice.
Reagents for the unified RCA protocol (Blocking → Hybridization → Ligation → RCA → Post-fix). Same for all sample types and readout technologies.
Enzymes & Inhibitors¶
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| SplintR Ligase (PBCV-1) | NEB | M0375S | 25 U/μl | Padlock probe ligation on RNA |
| Phi29 DNA Polymerase | Thermo Scientific | EP0091 | 10 U/μl | RCA; alt: NEB M0269S |
| RiboLock RNase Inhibitor | Thermo Scientific | EO0381 | 40 U/μl | Add to every reaction mix |
| RNase H (FFPE only) | NEB | M0297S | 5 U/μl | Add to blocking for FFPE samples |
Buffers¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| SplintR Reaction Buffer (10X) | NEB | (with M0375) | |
| Phi29 Reaction Buffer (10X) | Thermo Scientific | B62 | |
| Ampligase Reaction Buffer (10X) | Lucigen / Biosearch | (with Ampligase kit) | Blocking & hybridization |
Nucleotides & Oligos¶
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| dNTP Solution Mix | NEB | N0447S | 10 mM each | |
| Aminoallyl-dUTP | Thermo Scientific | R1101 | 50 mM | Cross-links rolonies |
| RCA Primer(s) | IDT (custom) | -- | 10 μM | |
| Oligo dT (blocking) | IDT (custom) | -- | 100 μM | |
| Padlock Probes | IDT (custom) | -- | panel-dependent | Experiment-specific design |
Blocking & Other¶
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| Recombinant Albumin (BSA) | NEB | B9000S | 20 μg/μl | |
| tRNA (yeast) | Invitrogen / Ambion | AM7119 | 10 μg/μl | |
| Glycerol | Any (mol. bio. grade) | -- | 100% | 5--10% in RCA |
| KCl | Any (mol. bio. grade) | -- | 1 M | |
| Formamide (deionized) | Sigma-Aldrich | F9037 | 100% | Hybridization & stripping |
| SSC (20X) | Any | -- | Stringent wash |
Autofluorescence Quenching (Optional)¶
When to order
Order if working with FFPE, or FF tissues with high autofluorescence. Sample-dependent — not always needed.
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Sudan Black B | Sigma-Aldrich | 199664 | 0.1% in 70% EtOH; filter before use |
Reagents specific to PRISM single-round fluorescence imaging readout.
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| PRISM Fluorescent Detection Probes | IDT (custom) | -- | Color-intensity barcoded; experiment-specific design |
Note
PRISM readout uses conventional fluorescence microscopy. No flow cell, pump, or sequencing reagents needed.
Reagents and equipment specific to SPRINTseq in-situ sequencing readout.
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Fluorescent reversible terminator nucleotides | (chemistry-dependent) | -- | Two-color SBS chemistry |
Additional Equipment¶
| Item | Notes |
|---|---|
| Flow Cell | Automated reagent delivery for sequencing cycles |
| Microfluidic Pump | Programmable; 100--200 μl/min |
| Syringe Filters (0.2--0.45 μm) | Pre-filter all reagents before flow cell |
Reagents for morphology staining performed after PRISM/SPRINTseq readout. Each stain is independent — order only what you need.
DAPI (Nuclear)¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| DAPI | Thermo Scientific | D1306 | 5 μg/ml working conc.; Ex/Em 358/461 nm |
Cell Membrane (WGA)¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| WGA-Alexa Fluor 488 | Thermo Scientific | W11261 | 5--10 μg/ml working; Ex/Em 495/519 nm; see protocol |
H&E¶
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| Mayer's Hematoxylin | Sigma-Aldrich | MHS16 | Pre-filter for flow cell use |
| Eosin Y (aqueous) | Sigma-Aldrich | HT110116 | Pre-filter for flow cell use |
| Scott's Tap Water Substitute | Sigma-Aldrich | S5134 | Bluing step |
| Acid Alcohol | -- | -- | 0.5% HCl in 70% EtOH; prepare fresh |
Immunofluorescence (Protein)¶
| Item | Notes |
|---|---|
| Primary antibodies | Target-specific; experiment-dependent |
| Secondary antibodies (fluorescent) | Species-matched; choose channels not overlapping with PRISM/SPRINTseq |
| Normal serum / BSA | Blocking for IF |
Core equipment needed across the workflow.
| Equipment | Spec | Used In |
|---|---|---|
| Cryostat | Blade -20°C, Head -10°C | FF pre-treatment (10 μm sections) |
| Hot Plate | 37°C | FF section adhesion |
| Water Bath | 80°C | FFPE RNA retrieval |
| Oven | 56--80°C | FFPE baking (56°C); PDMS curing (80°C) |
| Incubator / Thermostatic Block | 30--55°C range | RCA (30°C), Ligation (37°C), Hybridization (45--55°C) |
| Fluorescence Microscope | 20--40X, NA > 0.75; sCMOS | DAPI / FITC / Cy3 / Cy5 / Cy7 channels |
| Vacuum Desiccator + Pump | -- | PDMS degassing |
| Centrifuge | ≥10,000g | WGA stock clarification |
| -80°C Freezer | -- | Sample + enzyme storage |
| Microfluidic Pump | Programmable | SPRINTseq only |