PacBio Sequel II — Polymerase Binding and Optional Post‑Binding Pooling

Scope

Applies to size‑binned or single‑pool Iso‑Seq libraries constructed with SMRTbell Express v2.0 and, when applicable, barcoded overhang adapters. Instrument: Sequel II/IIe with SMRT Cell 8M.

Responsibilities

• Operator: Executes binding, optional pooling, loading, and run setup; records calculator outputs.

Safety

Follow institutional PPE and chemical hygiene. Handle SMRT Cells with clean, powder‑free gloves. Avoid contact with optical surfaces.

Materials and equipment

Reagents and kits

• Sequel II Binding Kit (e.g., Binding Kit 2.1) compatible with instrument chemistry.
• Complex Dilution Buffer (CDB) and other buffers supplied with the binding/sequencing kits.
• Nuclease‑free water.

Equipment and consumables

• Low‑bind tubes; calibrated pipettes; ice bucket.
• Vortex, microcentrifuge, and temperature‑controlled heat block.
• Clean bench area.

Inputs

• Libraries: SMRTbell Iso‑Seq libraries (e.g., Pool‑S ~1–3 kb and Pool‑L ~3.5–6 kb) with recorded concentrations (ng/µL) and mean sizes (bp).
• Molarity calculation: nM = (ng/µL × 10^6) / (660 × bp).

Planning and calculator setup

1. Open SMRT Link → Experiment Setup → Binding Calculator.
2. Chemistry: select the Sequel II Binding Kit version; Application: CCS/HiFi; Loading: Diffusion.
3. For each library (e.g., Pool‑S and Pool‑L), enter: mean insert size (bp), concentration, desired on‑plate concentration, movie time, and pre‑extension.
4. Record calculator outputs (volumes of library, polymerase, buffers; resulting complex concentration). Attach printout to the run plan.

Polymerase binding (perform per library)

Keep reagents on ice unless the IFU specifies a temperature. Thaw and mix all components gently. Use low‑bind plasticware.

1. Label tubes for each library (e.g., S and L).
2. Pipette library DNA, polymerase, and buffers exactly as specified by the Binding Calculator (Binding Kit 2.1 values vary by insert size and target pM).
3. Mix by gentle flick or brief vortex and quick‑spin.
4. Incubate for the time and temperature specified by the calculator/IFU.
5. Place bound‑complexes on ice. Do not freeze.

Acceptance (per library): Clear solution without precipitate; total bound‑complex volume sufficient for loading/optional pooling.

Optional post‑binding pooling (equimolar or weighted)

Pool after binding, as per the discussed plan. Default: equimolar S:L. If maximizing long isoforms, pool weighted (e.g., 60:40 L:S).

Equimolar pooling

• Compute molarity for each library before binding using measured concentration and mean size.
• Pool volumes proportional to molarity so that nM × volume is equal for each component. Example for two libraries: choose a convenient total (e.g., 100 µL) and set V_S = (nM_L / (nM_S + nM_L)) × 100 and V_L = (nM_S / (nM_S + nM_L)) × 100.
• Mix gently; keep on ice.

Weighted pooling

• Apply desired weights (e.g., L:S = 0.6:0.4) to the equimolar framework: effective nM_S = w_S × nM_S, effective nM_L = w_L × nM_L, then compute volumes as above.

Note: If calculator output provides final complex concentrations, you may pool to match complex molarity directly. Document the method used.

Records and forms

• Binding Calculator printout(s) for each library.
• Pooling worksheet showing molarity, volumes, and final ratios.

Troubleshooting

• Visible precipitate during binding: Gently warm to room temperature and invert to dissolve; verify kit temperatures and avoid over‑concentrating DNA.