PacBio Iso‑Seq Library Preparation — SMRTBell Ligation

1. Purpose

Construct PacBio SMRTbell libraries from amplified Iso‑Seq cDNA using SMRTbell Express Template Prep Kit v2.0 with barcoded overhang adapters (ALB). This SOP covers library construction only.

2. Scope

Applies to cDNA generated by Takara SMARTer or other validated Iso‑Seq cDNA methods. Covers single‑pool or dual size‑bin inputs. Excludes polymerase binding, sequencing, and downstream analysis.

3. Responsibilities

• Operator: Executes steps and records QC.
• Project Lead/PI: Confirms acceptance criteria and approves deviations.
• QA/Records: Maintains forms, lot tracking, and sign‑offs.

4. Safety

Wear lab coat, gloves, and eye protection. Handle ethanol, magnetic beads, and enzymes per SDS. Dispose of waste per institutional policy.

5. Materials and equipment

5.1 Reagents and kits

• PacBio SMRTbell Express Template Prep Kit v2.0 (all components).
• PacBio Barcoded Overhang Adapter set (e.g., Overhang Adapter v3; barcode plate such as “8A”).
• AMPure PB or Promega ProNex beads.
• Elution Buffer (EB) and fresh 80% ethanol.
• Nuclease‑free water.

5.2 Equipment and consumables

• Magnetic rack compatible with tube format.
• Thermal cycler or heat block with precise temperature control.
• Qubit (DNA HS assay) or equivalent fluorometer.
• Fragment analyzer (Femto Pulse, Bioanalyzer, or TapeStation).
• Low‑bind tubes, calibrated pipettes, microcentrifuge.

6. Input requirements

• Amplified cDNA mass per library: >160 ng and ≤500 ng (Sequel II workflows).
• Volume constraint: ≤ 47.4 µL cDNA entering Damage Repair.
• Concentration: ≥ 3.5 ng/µL recommended.
• Quality: Clean smear with minimal primer‑dimer; documented size distribution.

Pause points: Post‑ligated, cleaned SMRTbell libraries may be stored 4 °C ≤24 h or −20 °C longer. Avoid repeated freeze‑thaw.

7. Procedure

Perform steps per sample or per size‑bin. Keep reagents on ice unless temperatures are specified. Record all actual volumes, times, and lot numbers.

C1. DNA Damage Repair

1. In a low‑bind tube combine in order:

   • 7.0 µL DNA Prep Buffer
   • Up to 47.4 µL amplified cDNA (target mass >160–≤500 ng)
   • 0.6 µL NAD
   • 2.0 µL DNA Damage Repair Mix v2
   • Add nuclease‑free water to 57.0 µL total.

2. Mix gently, quick‑spin, and incubate 37 °C for 30 min.

C2. End Repair / A‑Tailing

1. Add 3.0 µL End Prep Enzyme Mix to reach 60.0 µL.
2. Incubate 20 °C for 30 min, then 65 °C for 30 min.
3. Brief spin; place on ice.

C3. Overhang Adapter Ligation (Adapter‑Ligation Barcodes)

1. Add the following to reach 95.0 µL:

   • 3.0 µL Overhang Adapter v3 (select barcode; record ID and sequence)
   • 30.0 µL Ligation Mix
   • 1.0 µL Ligation Enhancer
   • 1.0 µL Ligation Additive

2. Mix gently, quick‑spin, incubate 20 °C for 60 min.

3. Record barcode‑to‑sample map in the run plan.

C4. SMRTbell Cleanup (and Exonuclease, if applicable)

1. Add 95 µL magnetic beads (AMPure PB or ProNex). Mix thoroughly; incubate 5 min at room temperature.
2. Place on magnet until clear; remove supernatant.
3. Wash 2× with 200 µL 80% EtOH without disturbing beads.
4. Briefly air‑dry (do not overdry).
5. Elute with 12 µL EB at room temperature or 37 °C for 5 min; mix and place on magnet; transfer eluate to a new tube.

Note: Some kit revisions place an exonuclease digestion before cleanup. Follow IFU order if provided; document any deviations.

C5. Post‑ligation QC and release

1. Quantify using Qubit DNA HS.

2. Profile size distribution using Femto Pulse/Bioanalyzer/TapeStation.

3. Acceptance:

   • Distinct library peak at expected size.
   • Adapter‑dimer/short‑fragment fraction <5% by area.
   • Library mass available for downstream binding ≥160 ng (per library).

4. Label and store per pause‑point guidance.

8. Documentation and deliverables

• Lot tracking: Record kit name, version, and lot for each reagent.
• Barcode map: Table of sample → barcode ID and adapter sequence.
• QC records: Qubit readings, electropherograms, and notes on cleanups.
• Handoff manifest: Library ID, volume, concentration, calculated mass, expected insert mean/N50, barcode ID, storage conditions.

9. Troubleshooting

• Low ligation yield: Verify End Repair/A‑tail incubation times; check reagent age; confirm input mass within range.
• High adapter‑dimer: Reduce input below upper mass limit; ensure adequate bead cleanup; avoid bead overdrying; confirm adapter dilution.
• Poor recovery after cleanup: Warm EB to 37 °C and extend elution to 10 min; resuspend beads thoroughly before magnet.
• Skewed size profile: Confirm upstream cDNA size distribution; review bead cleanup ratios; check for partial overdrying.
• Barcode misassignment risk: Re‑check barcode IDs and sequences; include no‑barcode control where possible.