PCR-cDNA Barcoding sequencing library preparation (SQK-PCB111.24)

Overview & Kit Contents

This protocol is based on the Oxford Nanopore Technologies PCR-cDNA Barcoding Kit (SQK-PCB111.24), suitable for generating full-length barcoded cDNA sequencing libraries from total RNA.

Kit components

• VNP primer, SSP primer (strand-switching primer)
• Barcoded PCR primers (BC01–BC24)
• Rapid Adapter (RAP)
• Elution Buffer (EB)

Required reagents (user-supplied)

• Lambda Exonuclease
• NEBNext Quick Ligation Reaction Buffer
• T4 DNA Ligase 2M U/ml
• 10 mM dNTP solution
• LongAmp Hot Start Taq 2X Master Mix
• Maxima H Minus Reverse Transcriptase (200 U/ul) with 5x RT Buffer
• RNaseOUT, 40 U/ul
• USER (Uracil-Specific Excision Reagent) Enzyme
• Exonuclease I
• AMPure XP beads

Required equipment

• Thermal cycler, magnetic rack, Qubit fluorometer, vortex mixer, microcentrifuge

Input RNA requirements

• Input: 1–50 ng poly(A)+ mRNA or 50–100 ng total RNA (RIN >= 7 recommended)
• RNA should be DNase-treated if genomic DNA contamination is suspected
• Prepare RNA in 7.5 uL nuclease-free water
• Add 1 uL VNP primer (2 uM) and 1 uL 10 mM dNTPs
• Incubate at 65 C for 5 min, then snap-cool on ice for 1 min

Reverse transcription and strand switching (~90 min)

1. Prepare RT mix on ice (per reaction):
   • 4 uL 5x RT Buffer
   • 1 uL RNaseOUT (40 U/uL)
   • 1 uL nuclease-free water
   • 2 uL Strand-Switching Primer (SSP, 10 uM)
   • 1 uL Maxima H Minus RT (200 U/uL)
2. Add 10 uL RT mix to the 9.5 uL RNA/primer/dNTP mix (total 19.5 uL)
3. Incubate: 42 C for 90 min (RT + template switching), 85 C for 5 min (inactivation), hold 4 C
4. Add 1 uL RNase H, incubate 37 C for 10 min
5. Briefly centrifuge and place on ice

PCR amplification with barcoded primers (~30 min)

1. Prepare PCR reaction (50 uL total):
   • 20 uL cDNA from RT step
   • 25 uL LongAmp Taq 2X Master Mix
   • 1.5 uL Barcode Primer (BCXX, 10 uM)
   • 3.5 uL nuclease-free water
2. Cycling conditions:
   • 95 C, 3 min (initial denaturation)
   • 18 cycles: 95 C 15 s / 62 C 15 s / 65 C 5 min
   • 65 C, 6 min (final extension)
   • Hold at 4 C

Note: 18 cycles is suitable for 50-100 ng total RNA input. Reduce to 14-15 cycles for higher inputs.

Bead clean-up and quantification

1. Add 40 uL AMPure XP beads (0.8x ratio) to 50 uL PCR product
2. Incubate at RT for 5 min
3. Place on magnetic rack, wait for pellet to form
4. Wash 2x with 200 uL freshly prepared 80% ethanol
5. Air-dry pellet for ~30 s (do not over-dry)
6. Elute in 12 uL EB
7. Quantify with Qubit dsDNA HS assay
8. Expected yield: 50-500 ng depending on input RNA quality and quantity

Pool barcoded libraries and attach Rapid Adapter

1. Pool equimolar amounts of barcoded cDNA libraries (target: 100-200 fmol total)
2. Adjust volume to 11 uL with EB
3. Add 1 uL Rapid Adapter (RAP)
4. Mix gently by flicking, spin down briefly
5. Incubate at RT for 5 min
6. Library is ready for loading -- do not vortex after adapter attachment

Flow cell priming and loading

1. Prepare priming mix: 30 uL FCT + 1170 uL FCF
2. Load 800 uL priming mix via priming port, wait 5 min
3. Prepare loading mix: 12 uL adapted library + 37.5 uL SB + 25.5 uL LB
4. Load remaining priming mix, then load 75 uL library via SpotON port
5. Start sequencing in MinKNOW (Kit: SQK-PCB111.24, enable barcode demultiplexing)

References

• Oxford Nanopore Technologies, "PCR-cDNA Barcoding Kit (SQK-PCB111.24)".
• Earlier version: SQK-PCS111. Kit superseded by SQK-PCS114/SQK-PCB114 (V14 chemistry, R10.4.1 flow cells) in late 2023.