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.
For what a run of this costs -- per 100 μl reaction and per section -- see the Cost Model.
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 | P6887-250MG | ≥3,200 units/mg protein (datasheet). Prep and U/mL dosing: Pepsin Prep |
| HCl (conc.) | Any | -- | 12 M; dilute to 0.1 M — the single acid used for the pepsin stock, the pepsin working solutions, and acid-only FF permeabilization |
| 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) | -- | Forms the reaction chamber over the section. 2.5 x 2.5 cm block, ~5 mm thick, 5 mm punch ≈ 98 µl. See PDMS Chamber Fabrication |
| Microfluidic Hole Punch, 5 mm round | Generic (Taobao: 微流控打孔器) | -- | Punches the chamber cavity out of the cured PDMS sheet. Buy by diameter/shape, not part number; consumable — keep a spare. See PDMS Chamber Fabrication |
| Microscope Slides | Superfrost Plus | -- | |
| RNaseZap | Invitrogen | AM9780 | Decontaminate all surfaces |
Pepsin: order by activity, not by mass
P6887 is ≥3,200 U/mg protein; P7000 is ≥250 U/mg — a ~13-fold difference at the same mg/mL, so swapping catalogue number is not a substitution. Record the catalogue number and lot in the run log, and re-titrate whenever either changes. Concentrations are given in mg/mL throughout; older records use %(w/v), where 0.04% = 0.4 mg/mL.
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 |
| RVC (Ribonucleoside Vanadyl Complex) | NEB | S1402S | 200 mM, 10 mL, store −20 °C. Equimolar mix of the four ribonucleosides complexed with oxovanadium(IV). Dilute 1:20 (v/v) into the citrate retrieval buffer → 10 mM final, NEB's stated concentration for maximum RNase protection. First use: thaw sealed at 65 °C to a clear green-black solution, aliquot with minimal headspace, refreeze. Restored in pretreatment-ffpe v2.0 |
| Coverslips | Any | -- | FFPE section mounting |
| EasyDip Staining Jars | Any | -- | Decontaminate with RNaseZap |
NaBH₄ has moved out of pre-treatment
The pre-treatment quench was removed in pretreatment-ffpe v2.0 — quenching
runs after RCA. NaBH₄ is listed under Autofluorescence Quenching in the
Readout tab.
Reagents for the unified RCA protocol (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 (direct-RNA route) |
| Phi29 DNA Polymerase | Thermo Scientific | EP0091 | 10 U/μl | RCA; alt: NEB M0269S |
| RiboLock RNase Inhibitor | Thermo Scientific | EO0381 | 40 U/μl | Hybridization, ligation, RT. Not used in the RCA mix — the template is DNA by then |
Enzymes — cDNA route only¶
When to order
Only needed for the cDNA strategy. Skip if you are running direct RNA.
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| Maxima H Minus Reverse Transcriptase | Thermo Scientific | EP0751 | 200 U/μl | RNase H-minus — required so the RNA:cDNA hybrid survives until RNase H is added deliberately. Ships with 5X RT Buffer. 10 U/μl final |
| RNase H | NEB | M0297S | 5 U/μl | Digests the RNA strand before padlock hybridization |
| T4 DNA Ligase | NEB | M0202S | 5 U/μl | Default cDNA-route ligase. Not adequate for SNP calling |
| Exonuclease I | Thermo Scientific | EN0581 | 20 U/μl | Optional cleanup of unligated linear probes |
Enzymes — SNP / allele discrimination¶
T4 and Ampligase are not sufficient here
Single-nucleotide work needs a high-fidelity thermostable ligase run at 45--65°C. Either of the two below works — see Ligase Fidelity for the benchmark behind this.
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| Tth DNA Ligase | Thermo Scientific | EL0981 | -- | Best fidelity in the benchmark (7 single-mismatch mis-ligations, 0 on the 3' side); the historically more widely used choice |
| HiFi Taq DNA Ligase | NEB | M0647S | as supplied | 21 mis-ligations; ready-to-use, NAD⁺ already in its buffer. 2 μl per 100 μl, 45°C |
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) | Hybridization |
| 5X RT Buffer | Thermo Scientific | B91 (or with EP0751) | cDNA route. 5X, not 10X — 20 μl per 100 μl |
| T4 DNA Ligase Buffer (10X) | NEB | (with M0202) | Contains ATP |
| HiFi Taq DNA Ligase Buffer (10X) | NEB | (with M0647) | Contains 1 mM NAD⁺ at 1X. Store cold — NAD⁺ is what ages |
Nucleotides & Oligos¶
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| dNTP Solution Mix | NEB | N0447S | 10 mM each | 0.25 mM in RCA; double to 0.5 mM for low-yield samples (e.g. TNBC) |
| Aminoallyl-dUTP | Thermo Scientific | R1101 | 50 mM | Cross-links rolonies. Dilute to a 2 mM working stock for the RCA mix; scale with the dNTPs |
| RCA Primer(s) | IDT (custom) | -- | 10 μM | 0.1 μM final (1 μl per 100 μl) |
| Random Decamers | IDT (custom) | -- | 100 μM | cDNA route; 5 μM final |
| Custom RT Primer(s) | IDT (custom) | -- | 10 μM | cDNA route; 100 nM each, spiked in alongside the decamers to lift low-abundance transcripts |
| Padlock Probes | IDT (custom) | -- | panel-dependent | Experiment-specific design. 10 nM per probe for the preferred overnight hybridization |
Blockers & Other¶
| Reagent | Vendor | Cat # | Stock | Notes |
|---|---|---|---|---|
| Recombinant Albumin (BSA) | NEB | B9000S | 20 μg/μl | Blocker; hybridization, ligation and RCA mixes |
| tRNA (yeast) | Invitrogen / Ambion | AM7119 | 10 μg/μl | Carrier / blocking nucleic acid; hybridization mix. Not in the cDNA route's RT mix — it would give the RT an alternative template pool |
| 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. Applied after RCA; see Autofluorescence Quenching.
Order by which autofluorescence you have — these are not interchangeable
Lipofuscin (brain, aged, cardiac tissue) → Sudan Black B or TrueBlack. Erythrocyte / collagen / elastin (FFPE tumour sections) → TrueVIEW, which by the vendor's own statement does not quench lipofuscin. Aldehyde-fixation → NaBH₄ covers this destructively and cheaply.
| Reagent | Vendor | Cat # | Notes |
|---|---|---|---|
| NaBH₄ (Sodium Borohydride) | Sigma-Aldrich | 213462 | 1% in PBS, fresh on ice; run with strong-light photobleaching. Targets aldehyde AF. Also listed under Pre-treatment |
| Sudan Black B | Sigma-Aldrich | 199664 | 0.1% in 70% EtOH; filter before use. Targets lipofuscin / lipid AF |
| TrueBlack Lipofuscin Quencher (20X in DMF) | Biotium | 23007 | 1 mL, ~100–200 sections; dilute 1:20 in 70% EtOH. Heat stock to 70 °C / 5 min before use |
| TrueBlack Plus (40X in DMSO) | Biotium | 23014 | 500 μL; dilute 1:40 in PBS — aqueous, avoids the ethanol problem. Preferred variant to trial. 23014-T = 50 μL trial size |
| Vector TrueVIEW AF Quenching Kit | Vector Laboratories | SP-8400 | Reagents A+B+C (5 mL ea) + VECTASHIELD Vibrance mountant; ~100–150 sections; ~$173. SP-8500 = same with DAPI mountant |
| CUBIC-L (delipidation) | -- | -- | Optional lipid extraction run before Sudan Black B: 10 min RT, then PBST 3 x 10 min. Additive to SBB, not a replacement |
Purchasing caveats
- Biotium TrueBlack is special-order and the vendor publishes only price ranges ($169–254; Plus $64–320 USD), not per-SKU pricing. Confirm with the distributor before raising a PO.
- TrueVIEW has no standalone reagent SKU — the kit price includes a mounting medium you are then locked into (the vendor states substituting it "may dramatically affect the outcome"), and slides must be imaged within 48 h. Do not buy it for a cyclic/strippable readout.
- Biotium cat # 23012 and 23013 are different products (IF background suppressor, WB blocking buffer) — not autofluorescence quenchers.
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 mg/ml stock. Working: 10 ng/ml (1:500,000, via a 1:1000 then 1:500 serial dilution) — 5 μg/ml bleeds into the 480 nm channel. 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 |
| Microfluidic Hole Punch | 5 mm round | Punching PDMS reaction chambers |
| Centrifuge | ≥10,000g | WGA stock clarification |
| -80°C Freezer | -- | Sample + enzyme storage |
| Microfluidic Pump | Programmable | SPRINTseq only |