Oct 20, 2022

Public workspacePacBio Iso-Seq Preparation for Sequel II Systems V.1

DOI
dx.doi.org/10.17504/protocols.io.n92ld9wy9g5b/v1
  • 1University College London
christina.toomey
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DOI: dx.doi.org/10.17504/protocols.io.n92ld9wy9g5b/v1
Protocol Citation: Emil Gustavsson 2022. PacBio Iso-Seq Preparation for Sequel II Systems. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld9wy9g5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 14, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47283
Keywords: PacBio Iso-Seq preparation, cDNA synthesis, cDNA amplification, LD PCR, amplified cDNA purification, cDNA purification, ASAPCRN
Abstract
The Sequel Systems generate long reads that are well-suited for characterizing fulllength transcripts produced from high-quality RNA samples. This document describes a method to construct Iso-Seq SMRTbell® libraries for sequencing on both systems allowing detection of full-length transcripts.
This protocol describes how to perform PacBio targeted Iso-Seq
Attachments
Icon for PacBio_Iso-Seq.pdf
PacBio_Iso-Seq.pdf
221KB
Materials
  • SMARTer™ PCR cDNA Synthesis Kit
  • RNA
  • 3' Smart CDS Primer II A
  • Deionized H2O
  • 5X First-Strand Buffer
  • DTT
  • dNTP Mix
  • SMARTer II A Oligonucleotide
  • RNase Inhibitor
  • SMARTScribe Reverse Transcriptase
  • TE Buffer
  • 10X Advantage 2 PCR Buffer
  • 50X dNTP Mix
  • 5' PCR Primer II A
  • 50X Advantage 2 Polymerase Mix
  • 1.2% agarose/EtBr gel in 1X TAE buffer
  • 0.5 M EDTA
  • 1X AMPure PB beads
  • LoBind tube
  • 70% ethanol
  • cDNA
  • DNA Prep Buffer
  • NAD
  • DNA Damage Repair Mix v2
  • H2O
  • Overhang Adapter v3
  • Ligation Mix
  • Ligation Enhancer
  • Ligation Additive
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis

Note
This protocol has been optimized for both total RNA and poly A+ RNA. The minimum amount of starting material for cDNA synthesis is 2 ng of total RNA or 1 ng of poly A+ RNA. However, if your RNA sample is not limiting, we recommend that you start from 1 μg of total RNA or 0.5 μg of poly A+ RNA for cDNA synthesis.

For each sample and Control Mouse Liver Total RNA, combine the following reagents in separate 0.5 ml reaction tubes:

AB
1–3.5 μl RNA (1 ng–1 μg of poly A+ RNA or 2 ng–1 μg total RNA)
1 μl 3’ SMART CDS Primer II A (12 μM)
x μlDeionized H2O
4.5 μl Total Volume

Mix contents and spin the tubes briefly in a microcentrifuge.
Centrifigation
Mix
Incubate the tubes at Temperature72 °C in a hot-lid thermal cycler for Duration00:03:00 , then reduce the temperature to Temperature42 °C for Duration00:02:00 .

5m
Incubation
Prepare a Master Mix for all reaction tubes at room temperature by combining the following reagents in the order shown:

AB
2 μl 5X First-Strand Buffer
0.25 μl DTT (100 mM)
1 μl dNTP Mix (10 mM )
1 μl SMARTer II A Oligonucleotide (12 μM)
0.25 μlRNase Inhibitor
1 μl SMARTScribe Reverse Transcriptase (100 U)
5.5 μl Total Volume added per reaction

Aliquot Amount5.5 µL Master Mix into each reaction tube. Mix the contents of the tubes by gently pipetting and spin the tubes briefly to collect the contents at the bottom.

Centrifigation
Mix
Incubate the tubes at Temperature42 °C for Duration01:30:00 .

1h 30m
Incubation
Terminate the reaction by heating the tubes at Temperature70 °C for Duration00:10:00 .

10m
Dilute the first-strand reaction product by adding the appropriate volume of TE buffer (Concentration10 millimolar (mM) Tris [pH 8.0] , Concentration0.1 millimolar (mM) EDTA ):

Add Amount40 µL TE buffer if you used total RNA as the starting material.

Add Amount190 µL TE buffer if you used more than Amount0.2 µg poly A+ RNA as the starting material.

Add Amount90 µL TE buffer if you used less than Amount0.2 µg poly A+ RNA as the starting material.

cDNA Amplification by LD PCR
cDNA Amplification by LD PCR
For each sample and control, set up an extra reaction tube to determine the optimal number of PCR cycles. In our experience, each 100 μl reaction typically yields 1–3 μg of ds cDNA after the PCR and purification steps.

Table 1. Guidelines for Setting Up PCR Reactions

For each reaction, aliquot the appropriate volume (see Table I, above) of each diluted first-strand cDNA into a labeled 0.5 ml reaction tube. If necessary, add deionized H2O to adjust the volume to Amount10 µL .

Prepare a PCR Master Mix for all reactions, plus one additional reaction. Combine the following reagents in the order shown:

AB
74 μl Deionized H2O
10 μl 10X Advantage 2 PCR Buffer
2 μl 50X dNTP Mix (10 mM)
2 μl 5’ PCR Primer II A (12 μM)
2 μl 50X Advantage 2 Polymerase Mix
90 μl Total Volume per reaction

Mix well by vortexing and spin the tube briefly in a microcentrifuge.
Centrifigation
Mix
Aliquot Amount90 µL PCR Master Mix into each tube from Step 11.

Cap the tube. Commence thermal cycling using the following program:

• Temperature95 °C Duration00:01:00
• X number of cycles (consult Table 1).
• Temperature95 °C Duration00:00:15
• Temperature95 °C Duration00:00:30
• Temperature95 °C Duration00:03:00

4m 45s
PCR
Subject each tube from step 9 to 15 cycles, then pause the program. Transfer Amount30 µL from each tube to a second reaction tube labelled “Optimization”. Store the “Experimental” tubes at Temperature4 °C . Using the Tester PCR tube, determine the optimal number of PCR cycles:

Transfer Amount5 µL from the 15 cycle PCR reaction tube to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

Return the Optimization tubes to the thermal cycler. Run three additional cycles (for a total of 18) with the remaining Amount25 µL PCR mixture .

PCR
Transfer Amount5 µL from the 18 cycle PCR reaction tube to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

Run three additional cycles (for a total of 21) with the remaining Amount20 µL PCR mixture .

PCR
Transfer Amount5 µL from the 21 cycle PCR to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

Run three additional cycles (for a total of 24) with the remaining Amount15 µL PCR mixture .

PCR
Transfer Amount5 µL from the 24 cycle PCR to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

Run three additional cycles (for a total of 27) with the remaining Amount10 µL PCR mixture .

PCR
Electrophorese each Amount5 µL aliquot of the PCR reaction alongside Amount0.1 µg 1 kb DNA size markers on a 1.2% agarose/EtBr gel in 1X TAE buffer. Determine the optimal number of cycles required for each experimental and control sample.

Retrieve the 15 cycle Experimental PCR tubes from Temperature4 °C , return them to the thermal cycler, and subject them to additional cycles, if necessary, until you reach the optimal number.

When the cycling is completed, analyse a Amount5 µL sample of each PCR product alongside Amount0.1 µg 1 kb DNA size markers on a 1.2% agarose/EtBr gel in 1X TAE buffer. Compare your results to Figure 1 to confirm that your reactions were successful.
Figure 1. cDNA amplification by LD PCR

Add Amount2 µL 0.5 M EDTA to each tube to terminate the reaction.

Purification of Amplified cDNA
Purification of Amplified cDNA
Add 1X AMPure PB beads to the amplified cDNA.
Mix by tapping the LoBind tube until the sample is homogeneous.
Mix
Incubate at TemperatureRoom temperature for Duration00:10:00 .

10m
Incubation
Place on magnetic rack until solution clears. Remove and discard supernatant.
With the tube still on magnet, add Amount200 µL freshly prepared 70% ethanol to the tube containing beads plus DNA (1/2).

Wash
Remove and discard 70% ethanol (1/2).
Wash
With the tube still on magnet, add Amount200 µL freshly prepared 70% ethanol to the tube containing beads plus DNA (2/2).

Wash
Remove and discard 70% ethanol (2/2).
Wash
Let beads air dry for Duration00:01:00 .

1m
Add Amount27 µL EB and remove the tube from the magnet. Mix by tapping the tube until the sample is homogeneous. Then incubate at TemperatureRoom temperature for Duration00:02:00 .

2m
Incubation
Mix
Place back on magnet. When the solution clears, remove Amount25 µL supernatant into new 1.5 mL LoBind tube.

Determine concentration using Qubit device or similar quantification assay.
Run Amount1 µL sample on Agilent DNA 12000 chip according to manufacturer’s instructions.

The captured cDNA is now ready for SMRTbell library construction.
Repair DNA Damage
Repair DNA Damage
In a LoBind microcentrifuge tube, add the following reagents:
AB
X μl cDNA for 500 ng
7 μl DNA Prep Buffer
0.6 μl NAD
2 μl DNA Damage Repair Mix v2
Up to 57 μl H2O
57 μl Total Volume per reaction

Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
Pipetting
Mix
Place in a thermocycler and run the following program:
• Temperature37 °C Duration00:30:00
• Hold at Temperature4 °C

30m
PCR
End Repair/A-Tailing
End Repair/A-Tailing
In a LoBind microcentrifuge tube, add the following reagents:

AB
57 μlReaction Mix from previous step
3 μl End Prep Mix
60 μl Total Volume per reaction

Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
Pipetting
Mix
Place in a thermocycler and run the following program:
• Temperature20 °C Duration00:30:00
• Temperature65 °C Duration00:20:00
• Hold at Temperature4 °C

50m
PCR
Overhang Adapter Ligation
Overhang Adapter Ligation
Add the following directly to reaction mix from previous step:

AB
60 μl Reaction Mix from Previous Step
3 μl Overhang Adapter v3
30 μl Ligation Mix
1 μl Ligation Enhancer
1 μl Ligation Additive
95 μl Total Volume per reaction

Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
Pipetting
Mix
Place in a thermocycler and run the following program:
• Temperature20 °C Duration01:00:00
• Hold at Temperature4 °C

1h
PCR
Purification of cDNA
Purification of cDNA
Add 1X AMPure PB beads to the amplified cDNA.
Mix by tapping the LoBind tube until the sample is homogeneous.
Mix
Incubate at TemperatureRoom temperature for Duration00:10:00 .

10m
Incubation
Place on magnetic rack until solution clears. Remove and discard supernatant.
With the tube still on magnet, add Amount200 µL freshly prepared 70% ethanol to the tube containing beads plus DNA (1/2).

Wash
Remove and discard 70% ethanol (1/2).
Wash
With the tube still on magnet, add Amount200 µL freshly prepared 70% ethanol to the tube containing beads plus DNA (2/2).

Wash
Remove and discard 70% ethanol (2/2).
Wash
Remove ethanol.
Check for any remaining droplets in the tube. If droplets are present spin down down and place tube back on magnetic rack and pipette of any remaining ethanol.
Centrifigation
Pipetting
Let tube air dry for Duration00:01:00 .

1m
Add Amount30 µL EB and remove the tube from the magnet. Mix by tapping the tube until the sample is homogeneous. Then incubate at TemperatureRoom temperature for Duration00:02:00 .

2m
Incubation
Mix
Place back on magnet. When the solution clears, remove Amount30 µL supernatant into new 1.5 mL LoBind tube.

Purification of cDNA library
Purification of cDNA library
Perform two rounds of Ampure PB bead clean up in the "Purification of cDNA" section.
Prepare for Sequencing
Prepare for Sequencing
Follow the SMRT Link Sample Setup v8.0 (or higher) instructions for preparing the sample for sequencing on the Sequel II System.