PROTOCOL EXAMPLE - Nanopore · EXAMPLE PROTOCOL: nanooretechco: Page 3 ... and start the protocol...
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Page 1/4 nanoporetech.com Direct cDNA sequencing for the MinION TM device using SQK-DCS108 Flow Cell Number: DNA Samples: Before start checklist Direct cDNA Sequencing Kit (SQK-DCS108) NEB Blunt/TA Ligase Master Mix Notebook sleeptimer/update off AMPure XP beads SuperScript IV Reverse Transcriptase (118090010) 250 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 sample Add 250 ng RNA in ≤ 7.5 μl (x μl) Add 2.5 μl of VNP Add 1 μl 10 mM dNTPs Add (7.5 μl – x μl) NFW Mix by flicking the tube + spin down Incubate for 5 mins at 65 °C, snap cool on pre- chilled freezer block In a new tube, mix together: 4 μl SuperScript IV buffer 1 μl RNaseOUT 1 μl 100 mM DTT 2 μl SSP Mix by flicking the tube + spin down Add to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down Incubate for 2 mins at 42 °C Add 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 0.2 ml PCR tube 0.2 ml PCR tube 11 µl DNA LoBind 19 µl 0.2 ml PCR tube 20 µl 11 µl 8 µl 1 µl LongAmp Taq 2x Master Mix (e.g. NEB M0287) 10 mM dNTP solution (e.g. NEB N0447) Microfuge VNP SSP RiboShredder (Epicentre, RS12500) Add 1 μl RiboShredder Incubate for 10 mins at 37 °C Add 17 μl resuspended AMPure XP beads at RT Incubate on rotator for 5 minutes, spin down and pellet on magnet. Discard the supernatant Keep on magnet, wash 2x with 200 μl fresh 70% EtOH, do not disturb pellet Briefly spin down, replace on magnet, pipette off residual wash. Briefly allow to dry. Elute cDNA in 20 μl nuclease-free water Incubate on a rotator for 10 minutes at RT Pellet beads on a magnet, remove cDNA eluate and transfer to fresh DNA LoBind cDNA in NFW 17 µl 20 µl 1 µl Wash 2x 200 µl 1 µl EXAMPLE PROTOCOL
Transcript of PROTOCOL EXAMPLE - Nanopore · EXAMPLE PROTOCOL: nanooretechco: Page 3 ... and start the protocol...
Page 1/4nanoporetech.com
Direct cDNA sequencing for the MinIONTM device using SQK-DCS108
Flow Cell Number: DNA Samples:
Before start checklist
Direct cDNA Sequencing Kit (SQK-DCS108)
NEB Blunt/TA Ligase Master Mix
Notebook sleeptimer/update off
AMPure XP beads
SuperScript IV Reverse Transcriptase (118090010)
250 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 250 ng RNA in ≤ 7.5 μl (x μl)Add 2.5 μl of VNPAdd 1 μl 10 mM dNTPsAdd (7.5 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
0.2 ml PCR tube
0.2 ml PCR tube 11 µl
DNA LoBind 19 µl
0.2 ml PCRtube 20 µl
11 µl
8 µl
1 µl
LongAmp Taq 2x Master Mix (e.g. NEB M0287)
10 mM dNTP solution (e.g. NEB N0447)
Microfuge
VNP
SSP
RiboShredder (Epicentre, RS12500)
Add 1 μl RiboShredderIncubate for 10 mins at 37 °CAdd 17 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 20 μl nuclease-free waterIncubate on a rotator for 10 minutes at RTPellet beads on a magnet, remove cDNA eluate and transfer to fresh DNA LoBind
cDNA in NFW
17 µl
20 µl
1 µl
Wash 2x 200 µl
1 µl
EXAMPLE PROTOCOL
Page 2/4nanoporetech.com
Direct cDNA sequencing for the MinIONTM device using SQK-DCS108
Flow Cell Number: DNA Samples:
MASSFLOW INSTRUCTIONS NOTES/ OBSERVATIONS TIME/DATE
AMX1D
In a 0.2 ml PCR tube, mix together:25 μl 2x LongAmp Taq Master MIx2 μl PR2 primer20 μl reverse-transcribed cDNA sample 3 μl nuclease-free waterMix by flicking the tube + spin down
Incubate using the following protocol:1 min @ 94 °C (1 cycle) 1 mins @ 50 °C (1 cycle) 15 mins @ 65 °C (1 cycle) Hold @ 4 °C
Transfer to a fresh DNA LoBind tubeAdd 40 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 nuclease-free waterIncubate on a rotator for 10 minutes at RTPellet beads on a magnet, remove cDNA eluate and transfer to fresh DNA LoBindcDNA in NFW
17 µl
21 µl
1 µl
Wash 2x 200 µl
0.2 ml PCRtube 50 µl
1 µl
30 µl
1 μl Qubit fluorometer
Add from NEBNext Ultra II End-Repair / dA-tailing Module: 7 μl Ultra II End-Prep buffer3 μl Ultra II End-Prep enzyme mix20 μl nuclease-free waterMix by flicking the tube + spin downTransfer to 0.2 ml PCR tubeIncubate for at 20 °C for 5 minutes and 65 °C for 5 minutes
Transfer sample to fresh DNA LoBind tubeAdd 60 μl resuspended AMPure XP beads at RT and mix by pipettingIncubate on rotator for 5 minutes, spin down and pellet on magnet.Discard the supernatant.Keep 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 dryResuspend pellet in 30 μl Nuclease-free water, incubate at RT for 2 minutesPellet beads on a magnet, remove eluate of end-prepped DNA and transfer to fresh DNA LoBind tube
Adapter ligation. Mix by flicking between each addition: 30 μl end-prepped DNA20 μl Adapter Mix 1D50 μl NEB Blunt / TA Master MixMix gently by flicking the tube and spin down Incubate at RT for 10 minutesVortex AMPure XP beads to resuspendTransfer 40 μl of beads to DNA LoBind tubeMix by pipetting, incubate at RT on a rotator mixer for 5 mins Pellet beads on magnet and remove supernatantAdd 140 μl ABB to beads. Close tube lid, resuspend beads by flicking. Pellet beads on magnet and remove supernatant. Repeat.
ABB
60 µl
30 µl
Wash 2x 200 µl
DNA LoBind 20 µl
0.2 ml PCR tube 60 µl
40 µl
DNA LoBind 30 µl
DNA LoBind 100 µl
70 µl
40 µl
2xWash 140 µl
PR2
EXAMPLE PROTOCOL
Page 3/4nanoporetech.com
Direct cDNA sequencing for the MinIONTM device using SQK-DCS108
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 SpotON 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
Loading the prepared libraryAdd 75 μl of sample to the flow cell via the sample 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.
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
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 Nuclease-free water 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
priming port samplecover port cover
ELB
12 µl
DNA LoBind 12 µl Store on ice
EXAMPLE PROTOCOL
Page 4/4nanoporetech.com
Direct cDNA sequencing for the MinIONTM device using SQK-DCS108
Flow Cell Number: DNA Samples:
MASSFLOW INSTRUCTIONS NOTES/ OBSERVATIONS TIME/DATE
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 RT
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
EXAMPLE PROTOCOL
