PacBio Iso‑Seq cDNA Preparation — Reverse Transcription, Template Switching, and PCR Amplification and Bead-based Size Binning

SOP ID: ISOSEQ‑cDNA‑PREP‑PREQC Version: 1.0 Effective date: ☐☐/☐☐/☐☐☐☐ Author/Owner: ____________________ Approver(s): ____________________ Review cycle: 12 months

Scope

Applies to eukaryotic RNA samples using Takara SMARTer PCR cDNA Synthesis for cDNA generation.

Responsibilities

• Operator: Executes steps and records materials, lots, times, and yields.
• PI/Lead: Approves plan and any deviations.
• QA/Records: Verifies documentation completeness.

Safety

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

Materials and equipment

Reagents and kits

• SMARTer track: Takara SMARTer PCR cDNA Synthesis Kit (PT4097‑1 or validated successor).
• Cleanup: AMPure PB or Promega ProNex magnetic beads; fresh 80% ethanol; Elution Buffer (EB).
• General: Nuclease‑free water; low‑bind tubes.

Equipment

Thermal cycler with heated lid; magnetic rack; Qubit (DNA HS and RNA HS kits) or equivalent; microcentrifuge; calibrated pipettes.

Input requirements and planning

• RNA quality: RIN/RQN ≥7.0 recommended; ≥8.0 ideal; A260/280 ≈ 2.0; DNase treat upstream if needed.

• RNA mass:

  • Iso‑Seq Express: 300 ng recommended (per reaction) unless otherwise validated.
  • SMARTer: 2 ng–1 µg total RNA or 1–1,000 ng poly(A)+ RNA.

• Pause point: After post‑PCR bead cleanup (store 4 °C ≤24 h or −20 °C longer).

• Record: Sample IDs, kit/lots, operator initials, times, volumes, and pre/post‑cleanup volumes.

Detailed procedure — Takara SMARTer PCR cDNA

First‑strand cDNA synthesis

• In a 0.2 mL tube, mix: 1–3.5 µL RNA (2 ng–1 µg total; or 1–1,000 ng poly(A)+), 1 µL 3′ SMART CDS Primer II A, nuclease‑free water to 4.5 µL.
• Thermocycler: 72 °C 3 min, then 42 °C 2 min.
• Add master mix: 2 µL 5× First‑Strand Buffer, 0.25 µL DTT, 1 µL dNTPs (10 mM), 1 µL SMARTer II A Oligo (12 µM), 0.25 µL RNase inhibitor, 1 µL SMARTScribe RT → add 5.5 µL (final 10 µL).
• Incubate 42 °C for 60–90 min; 70 °C for 10 min. Proceed to LD‑PCR.

LD‑PCR amplification

• Set up 100 µL LD‑PCR with Advantage 2 polymerase per IFU.

• Default cycling guidance by RNA input (without QC laddering here):

  • 1 µg: 18–20 cycles
  • 250 ng: 18–20 cycles
  • 50 ng: 19–21 cycles
  • 10 ng: 21–23 cycles
  • 2 ng: 23–25 cycles

• Avoid over‑cycling. Final extension per IFU. Hold 4 °C.

Optional size‑binning via bead ratios (post‑PCR)

• Apply one‑step selection by adjusting bead volume to the 100 µL PCR:

  • 86 µL → Standard (~2 kb modal).
  • 95 µL → Short‑enriched (<2 kb).
  • 82 µL → Long‑enriched (>3 kb).

• Perform 5 min bind, magnet, 2× 200 µL 80% EtOH washes, elute 47 µL EB.

STOP POINT: Purified PCR cDNA is ready for QC prior to SMRTBell preparation. ** Quantify by Qubit DNA HS; check size profile by Femto Pulse/Bioanalyzer. Accept clean smear centered at design size; minimal primer‑dimers.

Records and forms (prep phase only)

• Sample & reagent log: Sample IDs, RNA QC values (RIN/A260‑280), input mass, kit names/versions/lots, operator initials, step timestamps.
• Process worksheet: Actual volumes used; cleanup bead ratios; elution volumes; storage location and time at pause point.
• Deviation log: Description, root cause, impact, corrective action, approval.