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PCR-cDNA sequencing for the MinIONTM device using SQK-PCS108

Flow Cell Number: DNA Samples:

Before start checklist

cDNA-PCR Sequencing Kit (SQK-PCS108)

NEB Blunt/TA Ligase Master Mix

Notebook sleeptimer/update off

AMPure XP beads

SuperScript IV Reverse Transcriptase (118090010)

50 ng PolyA+ RNA

Thermal cycler NEBNext Ultra II End-repair/dA-tailing module

Vortexer and Hula mixer Magnetic rack for bead separation Pre-chilled freezer pack for 0.2 ml tubes at -20 °C

Pipettes P2, P20, P100/200, P1000 Nuclease-free water (NFW) DNA LoBind Eppendorf tubes 1.5 ml

Pipette tips P2, P20, P100/200, P1000 10 mM Tris-HCl pH 8.5 (if used) 0.2 ml thin-walled PCR tubes

Freshly prepared 70% EtOH Platform QC completed on flow cell Latest versions of MinKNOW and Desktop Agent (check for updates)

MinION SpotON FLO-MIN107

MASSFLOW INSTRUCTIONS NOTES/ OBSERVATIONS TIME/DATE

Set up the RT reaction for each sampleAdd 50 ng RNA in ≤ 9 μl (x μl)Add 1 μl of VNPAdd 1 μl 10 mM dNTPsAdd (9 μl – x μl) NFWMix by flicking the tube + spin downIncubate for 5 mins at 65 °C, snap cool on pre-chilled freezer block

In a new tube, mix together:4 μl SuperScript IV buffer1 μl RNaseOUT1 μl 100 mM DTT2 μl SSPMix by flicking the tube + spin downAdd to the snap-cooled, annealed mRNA, mix by flicking the tube and spin downIncubate for 2 mins at 42 °CAdd 1 μl SuperScript IV enzyme (200 U / μl) Mix by pipetting + spin down

Set up amplification reaction:Reverse transcription 10 mins @ 50 °C (1 cycle) Strand switching 10 mins @ 42 °C (1 cycle) Heat inactivation 10 mins @ 80 °C (1 cycle) Hold @ 4 °C

Set up four PCR reactions, each containing 5 μl RNA: Add 25 μl LongAmp Taq 2x Master Mix Add 1.5 μl cPRM Add 18.5 μl NFWAdd 5 μl reverse-transcribed RNASet up amplification reaction:Initial denaturation 30 secs @ 95 °C (1 cycle) Denaturation 15 secs @ 95 °C (11-18 cycles) Annealing 15 secs @ 62 °C (11-18 cycles) Extension 50 secs/kb @ 65 °C (11-18 cycles) Final extension 6 mins @ 65 °C (1 cycle) Hold @ 4 °CPool the four 50 µl reactions into 200 µl

0.2 ml PCR tube

0.2 ml PCR tube 11 µl

DNA LoBind 19 µl

0.2 ml PCRtube 20 µl

0.2 ml PCR tube 50 µl

11 µl

0.2 ml PCR tube 5 µl 45 µl

8 µl

1 µl

LongAmp Taq 2x Master Mix (e.g. NEB M0287)

10 mM dNTP solution (e.g. NEB N0447)

Microfuge

pool 0.2 ml PCR tube 200 µl

VNP

SSP

cPRM

EXAMPLE PROTOCOL

Page 2/3nanoporetech.com

PCR-cDNA sequencing for the MinIONTM device using SQK-PCS108

Flow Cell Number: DNA Samples:

MASSFLOW INSTRUCTIONS NOTES/ OBSERVATIONS TIME/DATE

Purify the cDNA in a fresh DNA LoBind:Add 160 μl resuspended AMPure XP beads at RT Incubate on rotator for 5 minutes, spin down and pellet on magnet. Discard the supernatantKeep on magnet, wash 2x with 200 μl fresh 70% EtOH, do not disturb pelletBriefly spin down, replace on magnet, pipette off residual wash. Briefly allow to dry.Elute cDNA in 21 μl RABIncubate on a rotator for 10 minutes at RTPellet beads on a magnet, remove cDNA eluate and transfer to fresh DNA LoBind

1 μl Qubit fluorometer for each cDNA library. Assess the cDNA fragment length using an Agilent Bioanalyser or equivalent

Take 500-700 μg of amplified cDNA, and make up to 20 μl in RAB

Add 5 μl cAMX to the cDNA libraryMix by flicking the tube and spin downIncubate on rotator for 5 mins at RTSpin down briefly

DNA LoBind 20 µl

cDNA in NFW

DNA LoBind 25 µl

160 µl

21 µl

5 µl

1 µl

2xWash 200 µl

Vortex AMPure XP beads to resuspendTransfer 20 μl of beads to the cDNA libraryMix by pipetting, incubate at RT on a rotator mixer for 5 mins Pellet beads on magnet and remove supernatantAdd 140μl ABB to beads. Close tube lid, resuspend beads by flicking. Pellet beads on magnet and remove supernatant. Repeat.

ABB

Elute the adapted library (Pre-sequencing Mix):Resuspend pelleted beads in 12 μl ELB and incubate at RT for 10 minutesPellet beads on the magnet, remove the eluate and transfer to new DNA LoBind tube

20 µl

2xWash 140 µl

DNA LoBind 12 µl Store on ice

12 µl

Before start checklist

MinION™ connected to computer with SpotOn Flow Cell Desktop Agent set up RBF and library on ice

Platform QC completed (can be done in parallel to library prep) Run name set NFW at RT

Computer set up to run MinKNOW Prepared library > 4 ng / ul

Prepare the MinION for sequencing protocol :The platform QC should be run prior to library preparation beginningAssemble the MinION and MinION Flow CellSetup MinKNOW to run the Platform QC – name the run and start the protocol script – NC_Platform_QC.pyAllow the script to run to completion and the number of active pores are reported

Prime the flow cell ready for the library to be loaded when library preparation is completePrepare priming buffer:480 μl RBF520 μl Nuclease-free water

RBF

Priming and loading the library

RAB

cAMX

ELB

priming port samplecover port cover

EXAMPLE PROTOCOL

Page 3/3nanoporetech.com

PCR-cDNA sequencing for the MinIONTM device using SQK-PCS108

Flow Cell Number: DNA Samples:

MASSFLOW INSTRUCTIONS NOTES/ OBSERVATIONS TIME/DATE

Prime the flow cellOpen the priming port. Draw back a few μls of buffer to make sure there is continuous buffer flow from the priming port across the sensor array.Load 800μl of the priming buffer. Wait 5 minutesGently lift the sample port cover to make the sample port accessible. Load 200μl of the priming buffer through the sample port

Prepare the library for loading 35.0 μl RBF kept at RT25.5 μl LLB kept at RT12.0 μl Adapted and tethered library 2.5 μl Nuclease-free waterMix gently by pipetting

RBF

800 µl

DNA LoBind 75 µl

200 µl

35.0 µl

25.5 µl

2.5 µl12 µl

DNA LoBind

Starting the sequencing script in MinKNOW:Return to MinKNOW, name the run, select the NC_48Hr_ Lambda_control_exp_Run_FLO_MIN107_SQK-PCS108_plus_ Basecaller.py for live basecalling) using the start in the MinKNOW dialogue boxMinKNOW will report the number of pores available for sequencing before data collection begins. These may differ from those reported in the Platform QC.Allow the protocol to proceed until MinKNOW reports Finished Successfully. Use the Stop in the Control Panel to finish the protocol.Close down MinKNOW and disconnect the MinIONIf using Albacore for local basecalling, please refer to the instructions in Albacore basecalling software

Loading the prepared libraryAdd 75 μl of sample to the flow cell via the SpotON port in a dropwise fashion. Ensure each drop flows into the port before adding the next.Gently replace the port cover, making sure the bung enters the sample portClose the priming port cover and replace the MinION lid.

After sequencing checklistWash the flow cell and store at 4 °C, or complete the returns form in the Nanopore Community

Return reagents to the freezer Store MinION at RTEXAMPLE PROTOCOL