Preparing Iso-Seq® v2
libraries using SMRTbell®
prep kit 3.0

Page 1

Procedure & checklist

Before you begin
This procedure describes the workflow for constructing Iso-Seq libraries using SMRTbell prep kit 3.0 from RNA for
sequencing on PacBio® Sequel® II and Revio™ systems. For generating Kinnex™ libraries from Iso-Seq libraries, pleaserefer to the Kinnex full-length RNA protocol.
Overview
Samples

1–24

Workflow time

8 hours [for up to 24 samples]

RNA input
Quality / size distribution

RIN (RNA integrity number) ≥7.0

Quantity

Total RNA 300 ng per library (minimum concentration 43 ng/µL per library)cDNA Input
Quantity³50ng per library for 1 SMRT® Cell 8M; ³100ng per library for 1 Revio SMRT
Cell

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Page 2

Workflow1

Input RNA quality control
Safe stop

2

cDNA synthesis + cleanup

3

cDNA amplification + cleanup
Safe stop

4

Repair and A-tailing

5

Adapter ligation + cleanup
Safe stop

6

Nuclease treatment + cleanup© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-396-000 REV05 MAY2024

Page 3Required materials and equipment
RNA and DNA sizing
2100 Bioanalyzer instrument

Agilent Technologies G2939BA

RNA 6000 Nano kit

Agilent Technologies 5067-1511

DNA High Sensitivity DNA kit

Agilent Technologies 5067-4626

DNA quantitation
Qubit Fluorometer

Thermo Fisher Scientific Q33238

Qubit 1X dsDNA HS Assay kit

Thermo Fisher Scientific Q33230

cDNA synthesis and amplification
Iso-Seq® Express 2.0 Kit

PacBio® 103-071-500

SMRTbell® cleanup beads

PacBio® 102-158-300

Elution buffer (50 mL)

PacBio® 101-633-500

SMRTbell® library preparation
SMRTbell® prep kit 3.0

PacBio® 102-182-700

SMRTbell® barcoded adapter plate 3.0 (optional; for barcoding**)

PacBio® 102-009-200

200 Proof ethanol, molecular biology or ACS grade

Any major lab supplier (MLS)

Nuclease-free water, molecular biology grade

Any MLS (e.g., Sigma-Aldrich W4502)

8-channel pipettes – P20 & P200)

Any MLS

Single-channel pipette – P2, P10, P20, P100 or P200

Any MLS

0.2 mL 8-tube strips

USA Scientific TempAssure 1402-4708

Microcentrifuge

Any MLS

Magnetic separation rack compatible with 0.2 mL 8-tube strips

Any MLS (e.g., V&P Scientific, Inc. VP 772F4-1)

Thermal cycler compatible with 0.2 mL 8-tube strips

Any MLS© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-396-000 REV05 MAY2024

Page 4General best practices
Accurately pipette SMRTbell cleanup beads because small changes in volume can significantly alter the size
distribution of your sample.
Equilibrate the SMRTbell cleanup beads at room temperature for 30 mins prior to use.
The workflow takes ~8hr to complete. If a stop is necessary, refer to workflow for safe stopping points.

Safety precautions
Refer to the Safety Data Sheet (SDS) for information on reagent hazards and protocols for safe handling, use, storage,
and disposal.

Multiplexing best practices
Multiplexing can be achieved with one of the following methods.
1. Barcoded cDNA primers using Iso-Seq primers bc01–12 in step 3 of the protocol. To multiplex, use the Iso-Seq
cDNA amplification primer in combination with Iso-Seq primers bc01–12 to amplify samples. After SMRTbell
cleanup, Iso-Seq samples can be pooled and brought through a single SMRTbell prep kit 3.0 reaction. Eachbarcoded primer is sufficient for 2 reactions, with the Iso-Seq kit supporting a total of 24 reactions.
2. Barcoded adapters using SMRTbell Barcoded Adapter Plate 3.0. In this case, use barcoded adapters at step (5)“adapter ligation” in the workflow.
3. A combination of the above 2 approaches.

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Page 5

Workflow steps
1. Input RNA quality control
This protocol requires high-quality RNA. Prior to library preparation, evaluate the size distribution of the input RNA to
determine whether it is suitable for the protocol.

a

Step
1.1

Instructions
Measure the RNA Integrity Number (RIN) with an Agilent 2100 Bioanalyzer Instrument using theRNA 6000 Nano kit.
Proceed to the next step of the protocol if sample quality is acceptable:

1.2

RIN

Quality recommendations≥7.0

Recommended. Proceed to next step of the protocol.

<7.0

Increased library failure rates or reduced data quality.

SAFE STOPPING POINT – Store at -70°C or below

2. cDNA synthesis
2.1 cDNA synthesis
In this step, total RNA samples are converted to first-strand cDNA products.

a

Step
2.1.1

Instructions
Quick-spin the Iso-Seq RT enzyme mix in the microcentrifuge to collect liquid, then place on ice.
Thaw the following components at room temperature, briefly vortex to mix, then quick-spin tocollect liquid and place on ice.

2.1.2

Tube
color

Reagent

OrangeIso-Seq RT primer mix (103-104-000)

PurpleIso-Seq RT buffer (103-103-900)

Red

Iso-Seq cDNA PCR mix (103-104-200)

Green

Iso-Seq cDNA amplification primer (103-104-400)

Blue

Iso-Seq Express TSO 2.0 (103-104-300)

White

Iso-Seq primer barcodes 01 – 12* (the number of primers thawed will depend on the number ofsamples processed) 103-104-500 through 103-105-600*If processing only one sample, any of the 12 Iso-Seq barcoded primers can be used.

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Page 6

2.2 Primer annealing for first-strand synthesis

a

Step

Instructions
For each RNA sample to be processed, prepare reagent mix 1 on ice by adding the following
components to each tube in the PCR strip tube.
Tube
color

a
2.2.1

OrangeComponents

VolumeTotal RNA (300 ng)<7 µL

Iso-Seq RT primer mix

2 µL

Nuclease-free water

Up to 9 µL

Total volume

9 µL

2.2.2

Thoroughly mix by pipetting up and down 10 times.

2.2.3

Quick-spin the tube strip in a microcentrifuge to collect liquid.
Incubate in a thermal cycler with the following program. Set the lid temperature to 80°C.

2.2.4

Temperature

Time

70°C

5 min

20°C

hold

Proceed immediately to the next step

2.3 Reverse transcription and template switching

a

Step

Instructions
For each RNA sample, prepare reagent mix 2 on ice by adding the following components in theorder and volume listed below. Adjust component volumes for the number of samples being
prepared, plus 10% overage.

2.3.1

a

Tube color Components
PurpleYellow

2.3.2

VolumeIso-Seq RT buffer (vortex briefly before use)

5 µL

Nuclease-free Water

3 µL

Iso-Seq RT enzyme mix

2 µL

Total volume added per reaction

10 µL

Pipette-mix and quick-spin in a microcentrifuge to collect all liquid.
Add 10 µL of reaction mix 2 to the 9 µL from reaction mix 1 (Section 2.2) for a total volume of 19
µL.

2.3.3

a

Tube

Reagent

VolumePrevious

Reagent mix 1 from step
2.2

9 µL

Reagent mix 2

10 µL

Total volume added per reaction

19 µL© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-396-000 REV05 MAY2024

Page 7

2.3.4

Thoroughly mix by pipetting up and down 10 times and then quick-spin to collect all liquid.
Incubate in a thermocycler with the following program. Set the lid temperature to 52°C.

2.3.5

Temperature

Time

42°C

45 min20°C

Hold

Proceed immediately to the next step.
2.3.6

Remove the sample tube from the thermal cycler and add 2 µL of Iso-Seq template switch oligo
to the 19 µL reaction at room temperature for a total volume of 21 µL. Mix by pipetting up anddown 10 times and then quick-spin to collect all liquid from the sides of the tube.
Return sample tube to thermal cycler and incubate with the following program. Set the lidtemperature to 52°C.

2.3.7

Temperature

Time

42°C

15 min4°C

hold

2.4 1.3X SMRTbell bead cleanup

a

Step
2.4.1

Instructions
For each sample, add 29 µL of elution buffer to the 21 µL reverse transcription and templateswitching reaction (Section 2.3) for a total volume of 50 µL.

2.4.2

Add 65 µL of resuspended, room-temperature SMRTbell cleanup beads.

2.4.3

Mix beads by pipetting 10 times or until evenly distributed.

2.4.4.

Quick-spin strip tubes in a microcentrifuge to collect liquid.

2.4.5

Leave at room temperature for 10 minutes to allow DNA to bind the beads.

2.4.6

Place the strip tubes in a magnetic separation rack until the beads separate fully from thesolution.

2.4.7

Slowly pipette off the cleared supernatant without disturbing the beads. Discard thesupernatant.

2.4.8

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into thestrip tube. After 30 seconds, pipette off the 80% ethanol and discard.

2.4.9

Repeat the previous step.

2.4.10 Remove residual 80% ethanol:• Remove the strip tube from the magnetic separation rack.
• Quick-spin the strip tube in a microcentrifuge.

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Page 8• Place the strip tube back in a magnetic separation rack until beads separate fully from thesolution.
• Pipette off residual 80% ethanol and discard.
2.4.11 Remove the strip tube from the magnetic rack. Immediately add 21 µL of elution buffer to thestrip tube and resuspend the beads by pipetting 10 times or until evenly distributed.
2.4.12 Quick-spin the strip tube in a microcentrifuge to collect liquid.
2.4.13 Leave at room temperature for 5 minutes to elute the DNA.
2.4.14 Place the strip tube in a magnetic separation rack until the beads separate fully from thesolution.
2.4.15 Slowly pipette off the cleared supernatant without disturbing the beads. Transfer 21 µL of thesupernatant to a new strip tube. Discard the old strip tube with beads.
2.4.16 Proceed to the next step of the protocol.

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Page 93. cDNA amplification
First-strand cDNA products are PCR-amplified and barcoded using barcoded Iso-Seq primers at this step.

3.1 cDNA amplification

a

Step
3.1.1

Instructions
For each sample, prepare reaction mix 3 on ice by adding the following components in the orderand volume listed below. Adjust component volumes for the number of samples being prepared,
plus 10% overage. Pipette mix master mix. Iso-Seq primer bc01–12 will be added to eachsample individually and should not be added to the master mix.

a

3.1.2

Tube color

Components

VolumeRed

Iso-Seq cDNA PCR mix

25 µL

Green

Iso-Seq cDNA amplification primer

2 µL

Total volume

27 µL

On ice, add 27 µL of reaction mix 3 to the 21 µL of the eluted cDNA (from previous Section 2.4).
Add 2 µL of Iso-Seq primer barcode 01–12 for a total volume of 50 µL.

aTube color

Component

VolumeWhite

Iso-Seq primer barcode

2 µL

3.1.3

Thoroughly mix by pipetting up and down 10 times and then quick spin to collect all liquid.

3.1.4.

Run the thermal cycler program below with the lid temperature set to 105°C. Keep sample on ice
until thermal cycler lid has heated to 105°C.
PCR program
45 seconds at 98°C

1 cycle10 seconds at 98°C
15 seconds at 60°C
3 minutes at 72°C

12 cycles

5 minutes at 72°C
Hold at 4°C

SAFE STOPPING POINT -- Store at 4°C or -20°C for long-term storage© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-396-000 REV05 MAY2024

Page 10

3.2 Cleanup of amplified cDNA using 0.9X SMRTbell Cleanup beads

a

Step
3.2.1

Instructions
Add 45 μL (0.9x) of resuspended, room-temperature SMRTbell cleanup beads to the 50 μL of
cDNA amplified reaction from Section 3.1. The correct ratio of beads to sample is critical at this
step.

3.2.2

Mix beads by pipetting 10 times or until evenly distributed.

3.2.3

Quick-spin strip tubes in a microcentrifuge to collect liquid.

3.2.4

Leave at room temperature for 10 minutes to allow DNA to bind beads.

3.2.5

Place the strip tubes in a magnetic separation rack until beads separate fully from the solution.

3.2.6

Slowly pipette off the cleared supernatant without disturbing the beads. Discard thesupernatant.

3.2.7

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into thestrip tube. After 30 seconds, pipette off the 80% ethanol and discard.

3.2.8

Repeat the previous step.
Remove residual 80% ethanol:• Remove the strip tube from the magnetic separation rack.

3.2.9• Quick-spin the strip tube in a microcentrifuge.
• Place the strip tube back in a magnetic separation rack until beads separate fully fromthe solution.
• Pipette off residual 80% ethanol and discard.

3.2.10 Remove the strip tube from the magnetic rack. Immediately add 47 µL of elution buffer to thestrip tube and resuspend the beads by pipetting 10 times or until evenly distributed.
3.2.11 Quick-spin the strip tube in a microcentrifuge to collect liquid.
3.2.12 Leave at room temperature for 5 minutes to elute DNA.
3.2.13 Place the strip tube in a magnetic separation rack until the beads separate fully from thesolution.
3.2.14 Slowly pipette off the cleared supernatant without disturbing the beads. Transfer 47 µL of thesupernatant to a new strip tube. Discard the old strip tube with beads.
Recommended: Measure concentration and size distribution of each cDNA sample.
• Take a 1 µL aliquot from each strip tube. Dilute each aliquot with 4 µL of elution buffer.
3.2.15• Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit.
• Dilute 1:4 dilution further to 1.5 ng/µL based on the Qubit reading if needed.
• Run 1 µL on an Agilent Bioanalyzer using a High Sensitivity DNA kit.

3.2.16 The expected recovery after cDNA amplification SMRTbell clean-up is >200 ng. A minimum of
100ng of total cDNA is recommended to proceed with the SMRTbell prep kit 3.0 (Step 4).

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Page 113.3. Pooling barcoded cDNA (skip if not multiplexing)a Step3.3.1

Instructions

Using the concentration reading from the Qubit fluorometer, pool an equal mass of each barcoded
cDNA sample. Use the maximum total combined mass possible without exceeding 500 ng and notless than 100 ng in 46 μL. Store any remaining purified amplified, barcoded cDNA at 4°C for future
use.

3.3.2

Quick spin the tube strip in a microcentrifuge to collect liquid.

3.3.3

Proceed to next step of the protocol.

4. Repair and A-tailinga StepInstructions

Add the following components in the order and volume listed below to a new microcentrifuge
tube. Adjust component volumes for the number of samples being prepared, plus 10% overage.
For individual preps, add components directly to the sample from the previous step at the
specified volumes and skip RM1 steps.
Reaction Mix 1 (RM1)

4.1

a

Tube
color

Component

VolumePurpleRepair buffer

8 µL

Blue

End repair mix

4 µL

Green

DNA repair mix

2 µL

Total volume

14 µL

4.2

Thoroughly mix RM1 by pipetting 10 times.

4.3

Quick-spin RM1 in a microcentrifuge to collect liquid.

4.4

Add 14 µL of RM1 to each cDNA sample. Pipette mix 10 times and quick spin to collect liquid.
Total reaction volume should be 60 µL.
Incubate in a thermocycler with the following program. Set the lid temperature to 75°C.

4.4

4.5

Time

Temperature

Notes

30 min37°C

Repair

5 min

65°C

A-tailing

Hold

4°C

Proceed to the next step of the protocol.

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Page 12

5. Adapter ligation
5.1. Adapter ligationa StepInstructions

Add 4 µL of SMRTbell adapter (non-barcoded) to each sample from the previous step if not
5.1.1

multiplexing or if only using the cDNA amplification barcodes for multiplexing.
OR
Add 4 µL of a SMRTbell barcoded adapter 3.0 to each sample to further multiplex samples.
Add the following components in the order and volume listed below to a new microcentrifuge
tube. Adjust component volumes for the number of samples being prepared, plus 10% overage.
For individual preps, add components directly to each sample from the previous step in the orderand volume listed below, then skip RM2 steps.

5.1.2

Reaction Mix 2 (RM2)aTube color

Component

VolumeYellow

Ligation mix

30 µL

Red

Ligation enhancer

1 µL

Total volume

31 µL

5.1.3

Thoroughly mix RM2 by pipetting 10 times.

5.1.4

Quick-spin RM2 in a microcentrifuge to collect liquid.

5.1.5

Add 31 µL of RM2 to each sample from previous step. Pipette mix 10 times and quick spin tocollect liquid. Total volume should be 95 µL.
Incubate in a thermocycler with the following program. Set the lid temperature to 75°C.

5.1.6

5.1.7

Time

Temperature

Notes

30 min20°C

Ligation

Hold

4°C

Proceed to the next step of the protocol.

© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 135.2. 1.3X SMRTbell bead cleanup

a

Step

Instructions

5.2.1

Add 124 µL of resuspended, room-temperature SMRTbell cleanup beads to each sample.

5.1.2

Mix beads by pipetting 10 times or until evenly distributed.

5.1.3

Quick spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.

5.1.4

Leave at room temperature for 10 minutes to allow DNA to bind beads.

5.1.5

Place tube strip in a magnetic separation rack until beads separate fully from the solution.

5.1.6

Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.

5.1.7

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into eachtube. After 30 seconds, pipette off the 80% ethanol and discard.

5.1.8

Repeat the previous step.
Remove residual 80% ethanol:• Remove tube strip from the magnetic separation rack.

5.1.9• Quick spin tube strip in a microcentrifuge.
• Place tube strip back in a magnetic separation rack until beads separate fully from thesolution.
• Pipette off residual 80% ethanol and discard.

5.1.10

Remove tube strip from the magnetic rack. Immediately add 40 µL of elution buffer to each tube
and resuspend the beads by pipetting 10 times or until evenly distributed.

5.1.11

Quick spin the tube strip in a microcentrifuge.

5.1.12

Leave at room temperature for 5 minutes to elute DNA.

5.1.13

Place tube strip in a magnetic separation rack until beads separate fully from the solution.

5.1.14

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a
new tube strip. Discard old tube strip with beads.

5.1.15

Proceed to the next step of the protocol.
SAFE STOPPING POINT – Store at 4°C© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 14

6. Nuclease treatment
6.1. Nuclease treatmenta StepInstructions

Add the following components in the order and volume listed below to a new microcentrifuge
tube. Adjust component volumes for the number of samples being prepared, plus 10% overage.
For individual preps, add components directly to each sample from the previous step in the orderand volume listed below, then skip RM3 steps.
6.1.1

Reaction Mix 3 (RM3)aTube color

Component

VolumeLight purple

Nuclease buffer

5 µL

Light green

Nuclease mix

5 µL

Total volume

10 µL

6.1.2

Thoroughly mix RM3 by pipetting 10 times.

6.1.3

Quick spin RM3 in a microcentrifuge to collect liquid.

6.1.4

Add 10 µL of RM3 to each sample. Pipette mix 10 times and quick spin to collect liquid. Total
volume should equal 50 µL.
Incubate reaction in a thermocycler with the following program. Set the lid temperature to 75°C.

6.1.5

6.1.6

Time

Temperature

Notes

15 min37°C

Nuclease treatment

Hold

4°C

Proceed to the next step of the protocol.

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102-396-000 REV05 MAY2024

Page 15

6.2. 1.3X SMRTbell bead cleanupa StepInstructions

6.2.1

Add 65 µL of resuspended, room-temperature SMRTbell cleanup beads to each sample.

6.2.2

Mix beads by pipetting 10 times or until evenly distributed.

6.2.3

Quick spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.

6.2.4

Leave at room temperature for 10 minutes to allow DNA to bind beads.

6.2.5

Place tube strip in a magnetic separation rack until beads separate fully from the solution.

6.2.6

Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.

6.2.7
6.2.8

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into eachtube. After 30 seconds, pipette off the 80% ethanol and discard.
Repeat the previous step.
Remove residual 80% ethanol:• Remove tube strip from the magnetic separation rack.

6.2.9• Quick spin tube strip in a microcentrifuge.
• Place tube strip back in a magnetic separation rack until beads separate fully from thesolution.
• Pipette off residual 80% ethanol and discard.

6.2.10

Remove tube strip from the magnetic rack. Immediately add 15 µL of elution buffer to each tube
and resuspend the beads by pipetting 10 times or until evenly distributed.

6.2.11

Quick spin the tube strip in a microcentrifuge.

6.2.12

Leave at room temperature for 5 minutes to elute DNA.

6.2.13

Place tube strip in a magnetic separation rack until beads separate fully from the solution.

6.2.14

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a
new tube strip. Discard old tube strip with beads.
Measure concentration and size distribution of each cDNA sample.
Take a 1 µL aliquot from each strip tube. Dilute each aliquot with 4 µL of elution buffer.

6.2.15

Measure DNA concentration with a Qubit Fluorometer using the 1x dsDNA HS kit.
Dilute sample further to 1.5 ng/µL based on the Qubit reading.
Run 1 µL on an Agilent Bioanalyzer using a High Sensitivity DNA kit.

6.2.16Store SMRTbell libraries at 4°C if sequencing within 2 weeks of prep. Otherwise, place libraries at 20°C for long-term storage. Minimize freeze-thaw cycles when working with libraries.
PROTOCOL COMPLETE© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 16

Revision history (description)

Version

Date

Initial release01

April 2022

Adjusted lid temperature, in step 5.1.6, to 75°C.

02

April 2022

Modified cDNA amplification PCR cycles to 12 for monomer, changed cDNA
amplification post-cleanup elution volume.

03

December 2023

Minor updates throughout

04

March 2024

Minor updates throughout

05

May 2024

Research use only. Not for use in diagnostic procedures. © 2024 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved.
Information in this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in thisdocument. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party
products. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific
Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, Onso, Apton,
Kinnex, and PureTarget are trademarks of PacBio.
© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-396-000 REV05 MAY2024