TITLE: PCR-cDNA Barcoding (SQK-PCB109)
AUTHORS: Oxford Nanopore Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.</div><div class = "text-block">The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, from an input of as low as 1 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.</div><div class = "text-block">Taking full-length poly-A+ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together, and Rapid Sequencing Adapters are added to the pooled mix.</div></div>

[STEPS]
?. [Reverse transcription and strand-switching]
Mix gently by flicking the tube, and spin down.
?. [Reverse transcription and strand-switching]
Prepare the following reaction in a 0.2 ml PCR tube:
?. [Reverse transcription and strand-switching]
Incubate at 65° C for 5 minutes and then snap cool on a pre-chilled freezer block.
?. [Reverse transcription and strand-switching]
In a separate tube, mix together the following: 4 μl 5x RT Buffer
?. [Reverse transcription and strand-switching]
Mix gently by flicking the tube, and spin down.
?. [Reverse transcription and strand-switching]
Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down.
?. [Reverse transcription and strand-switching]
Incubate at 42° C for 2 minutes.
?. [Reverse transcription and strand-switching]
Add 1 μl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 μl.
?. [Reverse transcription and strand-switching]
Mix gently by flicking the tube, and spin down.
?. [Reverse transcription and strand-switching]
Incubate using the following protocol:Reverse transcription and strand-switching 90 mins @ 42° C (1 cycle) Heat inactivation 5 mins @ 85° C (1 cycle)Hold @ 4° C
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
The PCR steps outlined below adds barcodes to each reverse transcribed RNA (cDNA) sample. The Barcode Primers provided in the PCR-cDNA Barcoding kit (SQK-PCB109) are used to barcode/multiplex up to 12 individual samples on a single flow cell.It is recommended that any remaining reverse transcription reaction is retained to allow for further PCR reactions if greater yield is required.
IMPORTANT: Each PCR reaction uses 5 μl of reverse transcribed RNA sample (out of a total of 20 μl). Therefore, sufficient material is available to perform up to four PCR reactions per sample. Do NOT, however, use all 20 μl of reverse transcribed RNA (cDNA) in a single PCR reaction.
IMPORTANT: This kit enables multiplexing of up to 12 samples. The default method allows you to perform one 50 μl PCR reaction per sample. If multiplexing two or three samples, however, two separate PCR reactions per sample should be performed; if running just one sample, four separate PCR reactions should be performed. These recommendations aim to ensure that enough PCR product is generated for optimal flow cell performance.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
For each sample (up to 12), prepare the following reaction at RT:
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Amplify using the following cycling conditions:
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Add 1 μl of NEB Exonuclease 1 (20 units) directly to each PCR tube. Mix by pipetting.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Incubate the reaction at 37° C for 15 min, followed by 80° C for 15 minutes.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Pool any PCR reactions containing the same barcoded sample in a clean 1.5 ml Eppendorf DNA LoBind tube.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Resuspend the AMPure XP beads by vortexing.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Add 0.8X equivalents of resuspended AMPure XP beads to the reaction and mix by pipetting.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Prepare 500 μl of fresh 70% ethanol in Nuclease-free water.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Keep the tube on the magnet and wash the beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Repeat the previous step.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Remove the tube from the magnetic rack and resuspend pellet in 12 μl of Elution Buffer (EB). Incubate on a Hula mixer (rotator mixer) for 10 minutes at RT.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Pellet beads on magnet until the eluate is clear and colourless.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Remove and retain 12 μl of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube.Dispose of the pelleted beads
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Analyse 1 μl of the amplified DNA for size, quantity and quality.
IMPORTANT: Sometimes a high-molecular weight product is visible in the wells of the gel when the PCR products are run, instead of the expected smear. These libraries are typically associated with poor sequencing performance. We have found that repeating the PCR with fewer cycles can remedy this.
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
In a 1.5 ml Eppendorf DNA LoBind tube, pool together a total of 100 fmol of the amplified cDNA barcoded samples to a final volume of 11 μl in Elution Buffer (EB).Please check the Mass to Molarity table in the protocol.
?. [Adapter addition]
Add 1 μl of Rapid Adapter (RAP) to the amplified cDNA library.
?. [Adapter addition]
Mix by pipetting and spin down.
?. [Adapter addition]
Incubate the reaction for 5 minutes at RT.
?. [Adapter addition]
Spin down briefly.
?. [Priming and loading the SpotON flow cell.]
The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.
Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol. It is provided in the kit for potential future product compatibility.
?. [Priming and loading the SpotON flow cell.]
Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at RT before placing the tubes on ice as soon as thawing is complete.
?. [Priming and loading the SpotON flow cell.]
Mix the Sequencing Buffer (SQB), Flush Buffer (FB) and Flush Tether (FLT) tubes by vortexing, spin down then return to ice.
?. [Priming and loading the SpotON flow cell.]
Open the MinION Mk1B lid and slide the flow cell under the clip.
?. [Priming and loading the SpotON flow cell.]
Slide the priming port cover clockwise to open the priming port.
IMPORTANT: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
?. [Priming and loading the SpotON flow cell.]
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):
?. [Priming and loading the SpotON flow cell.]
Prepare the flow cell priming mix: add 30 μl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing.
?. [Priming and loading the SpotON flow cell.]
Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.
?. [Priming and loading the SpotON flow cell.]
Thoroughly mix the contents of the Loading Beads (LB) by pipetting.
IMPORTANT: The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
?. [Priming and loading the SpotON flow cell.]
In a new tube, prepare the library for loading as follows:
?. [Priming and loading the SpotON flow cell.]
Complete the flow cell priming:
?. [Ending the experiment]
After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit instructions and store the washed flow cell at 2-8°C, OR
?. [Ending the experiment]
Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.
IMPORTANT: If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.
?. [Reverse transcription and strand-switching]
x μl 1 ng PolyA+ RNA (or ~50 ng total RNA)
?. [Reverse transcription and strand-switching]
1 μl VN Primers (VNP), at 2 μM
?. [Reverse transcription and strand-switching]
1 μl 10 mM dNTPs
?. [Reverse transcription and strand-switching]
9-x μl RNase-free water.
?. [Reverse transcription and strand-switching]
1 μl RNaseOUT
?. [Reverse transcription and strand-switching]
1 μl Nuclease-free water
?. [Reverse transcription and strand-switching]
2 μl Strand-Switching Primer (SSP, at 10 μM)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
5 μl Reverse-transcribed RNA sample
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
1.5 μl Barcode Primers (BP01-BP12)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
18.5 μl Nuclease-free water
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
25 μl 2x LongAmp Taq Master Mix
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Initial denaturation 30 secs @ 95° C (1 cycle)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Denaturation 15 secs @ 95° C (11-18* cycles)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Annealing 15 secs @ 62° C (11-18* cycles)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Extension 50 secs per kb @ 65° C (11-18* cycles)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Final extension 6 mins @ 65° C (1 cycle)
?. [Selecting for full-length transcripts by PCR and barcoding samples.]
Hold @ 4° C
?. [Priming and loading the SpotON flow cell.]
Set a P1000 pipette to 200 μl
?. [Priming and loading the SpotON flow cell.]
Insert the tip into the priming port
?. [Priming and loading the SpotON flow cell.]
Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.
?. [Priming and loading the SpotON flow cell.]
37.5 μl Sequencing Buffer (SQB)
?. [Priming and loading the SpotON flow cell.]
25.5 μl Loading Beads (LB), mixed immediately before use.
?. [Priming and loading the SpotON flow cell.]
12 μl DNA library
?. [Priming and loading the SpotON flow cell.]
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
?. [Priming and loading the SpotON flow cell.]
Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
?. [Priming and loading the SpotON flow cell.]
Mix the prepared library gently by pipetting up and down just prior to loading.
?. [Priming and loading the SpotON flow cell.]
Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
?. [Priming and loading the SpotON flow cell.]
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION Mk1B lid.