Jun 04, 2026

4. PacBio SMRTbell Library Prep 3.0 (Manual Version)

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpj4z8gzp/v1
  • 1CFSAN/FDA;
  • 2USDA;
  • 3US FDA
Jae Hee Jang
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpj4z8gzp/v1
External link: https://www.pacb.com/wp-content/uploads/Procedure-checklist-Preparing-whole-genome-and-metagenome-libraries-using-SMRTbell-prep-kit-3.0.pdf
Protocol Citation: Jae Hee Jang, Kathryn Judy, Ellie Meeks, Maria Hoffmann 2026. 4. PacBio SMRTbell Library Prep 3.0 (Manual Version). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpj4z8gzp/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: May 27, 2023
Last Modified: June 04, 2026
Protocol  Integer ID: 82537
Keywords: PacBio, Pacific Biosciences, SMRTbell, Long read, Library, Prep, 3.0, manual workflow for smrtbell library prep, pacbio smrtbell library prep, smrtbell prep kit, shearing 1x smrtbell bead cleanup, smrtbell library prep, using pacbio, fragment size of the dna input, dna input, kb for optimal assembly result, pacbio, sample, optimal assembly result
Abstract
This protocol outlines the manual workflow for SMRTbell library prep using PacBio's SMRTbell Prep Kit 3.0. It is recommended to use this protocol after "Post-Shearing 1x SMRTbell Bead Cleanup (Manual Version)". Ideally, the fragment size of the DNA input should be around 12-15 kb for optimal assembly results. The concentration of the samples should be at least 8 ng/µL (at least 300 ng total).
Guidelines
DNA input:
  • Single or pooled library: 1 µg per SMRT® Cell 8M
  • Multiplex libraries: 300 ng – 1 µg per sample

DNA quality:
  • Microbial and metagenomic samples: 90% ≥7 kb, but preferably around 15 kb for easier assembly

Reagent and sample handling
  • Room temperature is defined as any temperature in the range of 18–23°C for this protocol.
  • Thaw the repair buffer, nuclease buffer, and elution buffer at room temperature.
  • Mix reagent buffers with a brief vortex prior to use. Do not vortex enzymes.
  • Quick-spin all reagents in microcentrifuge to collect liquid at bottom prior to use.
  • Keep all temperature-sensitive reagents on ice.

  • Pipette-mix all bead binding and elution steps until beads are distributed evenly in solution.
  • It is recommended to pipette-mix all SMRTbell prep reactions by pipetting up and down 10 times.
  • Samples can be stored at 4°C at all safe stopping points listed in the protocol.

Pooling libraries with different fragment length profiles
  • Use the Express Microbial Multiplexing Calculator and input individual libraries' concentrations and fragment sizes to determine how much of each library to use.
Materials
Reagents
  • SMRTbell Prep Kit 3.0 (PacBio Cat# 102-182-700)
  • any of SMRTbell Adapter Index Plates (PacBio)
Plate 96A, Cat# 102-009-200
Plate 96B, Cat# 102-547-800
Plate 96C, Cat# 102-547-900
Plate 96D, Cat# 102-548-000
  • 80% Ethanol, Molecular Biology Grade, (Thermo Fisher Scientific Cat# T08204K7 or equivalent)
  • Qubit 1x dsDNA High Sensitivity Kit, (Thermo Fisher Scientific Cat# Q33231 or Q33230)

Supplies
  • Pipette Tips, Sterile, Filtered (P20, P200), any manufacturer
  • Wide-bore Pipette Tips, Sterile, Filtered (P200) (Mettler Toledo Cat# 30389241 or equivalent)
  • PCR 8-tube strips, (USA Scientific Cat# 1402-4700 or equivalent)
  • Qubit Assay Tubes or Qubit Flex Assay Tube Strips, (Thermo Fisher Scientific Cat# Q32856 or Q33252)
  • 1.5 mL PCR-clean, low DNA binding microfuge tubes (Eppendorf DNA LoBind Cat# 0030108418 or equivalent)
  • optional Reagent Reservoirs, (Thermo Fisher Scientific Cat# 13681504 or equivalent)

Equipment
  • Micropipettes, single (P2, P20, P200) and multichannel (P20, P200), any manufacturer
  • Qubit Fluorometer (4.0, 3.0, or 2.0) or Qubit Flex Fluorometer (Thermo Fisher Scientific Cat# Q33238 or Q33327)
  • Themocycler with heated lid, (Bio-Rad Cat# 1861096 or equivalent)
  • Microcentrifuge (Benchmark Scientific Cat# C1012 or equivalent)
  • Vortex (Benchmark Scientific Cat# BV101-B or equivalent)
  • Magnetic separation rack compatible with 0.2 mL 8-tube strips, any manufacturer
  • Magnetic separation rack compatible with 1.5 mL Eppendorf tubes, any manufacturer
  • Ice bucket, any manufacturer
Safety warnings

Safety information
Chemical Safety Warning: Take proper precautions and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.

PacBio SMRTbell Prep Kit 3.0: See PacBio SDSs for more information. No components of the SMRTbell Prep Kit 3.0 are classified as hazardous substances or mixtures.

Before start
  • Samples should be free of RNA before beginning library prep. If RNA is present, treat with RNase A and incubate at 37C for 15 minutes. Clean reactions using a 1X concentration of SMRTbell cleanup beads (or AMPure PB) before proceeding. RNA can reduce loading of your library on the SMRT Cell if not removed prior to library prep.
  • Program thermocycler(s) prior to beginning the protocol for the first time.
Repair and A-tailing
50m
Add the following components in the order and volume listed below to a new microcentrifuge tube
  • Adjust component volumes for the number of libraries being prepared, plus 10% overag
  • If preparing 8 or more libraries, increase the overage to 25% to ensure there is enough reaction mix to dispense
  • For individual preps, add components directly to the sample from the previous step at the specified volumes and skip RM1 steps (2 to 4)


Figure 1: Purple reagent is Repair buffer and needs 8µL per library. Blue reagent is End Repair mix and needs 4µL per library. Green reagent is DNA repair mix and needs 2µL per library. This is a total of 14 µL per library, plus an additional 10% to account for pipetting error
Pipette-mix RM1
Quick-spin RM1 in a microcentrifuge to collect liquid
Add 14 µL of the RM1 to each sample. Total reaction volume should be 60 µL
Pipette-mix each sample using wide-bore pipette tips
Quick-spin the tube strip in a microcentrifuge to collect liquid
Run the repair and A-tailing thermocycler program

TimeTemperature
1 hr37°C
10 min65°C
Hold4°C

Proceed to the next step of the protocol
Adapter ligation
50m
If barcoding samples: add 4 µL of barcoded adapters from the SMRTbell barcoded adapter plate 3.0 to each respective sample from the previous step and exclude the SMRTbell adapter from Reaction Mix 2. Skip this step if not barcoding samples
Add the following components in the order and volume listed below to a new microcentrifuge tube.
  • Adjust component volumes for the number of libraries being prepared, plus 10% overage
  • If preparing 8 or more libraries, increase the overage to 25% to ensure there is enough reaction mix to dispense
  • For individual preps, add components directly to each sample from the previous step in the order and volume listed below, then skip RM2 steps (11 to 13)
  • *Exclude the SMRTbell adapter if using the SMRTbell barcoded adapter plate 3.0.

Figure 2: Orange reagent is SMRTbell adapter and needs 4 µL per library only for non-barcoded samples. Yellow reagent is Ligation mix and needs 30 µL per library. Red reagent is Ligation enhancer and needs 1 µL per library. This is a total of 35 µL per library for non-barcoded samples, or 31 µL for barcoded samples, plus an additional 10% to account for pipetting error
Pipette-mix RM2
Quick-spin RM2 in a microcentrifuge to collect liquid
Barcoded samples: add 31 µL of RM2 to each sample from the previous step.
Non-barcoded samples: add 35 µL of RM2 to each sample from the previous step.

Total volume per sample should be 95 µL
Pipette-mix each sample using wide-bore pipette tips
Quick-spin the tube strip in a microcentrifuge to collect liquid
Run the adapter ligation thermocycler program
TimeTemperature
30 min20°C
Hold4°C

Cleanup with 1X SMRTbell cleanup beads
1h
Add 95 µL of resuspended, room-temperature SMRTbell cleanup beads to each sample
Using wide-bore pipette tips, pipette-mix the beads until evenly distributed
Quick-spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes
Leave at Room temperature for 00:10:00 to allow DNA to bind beads
10m
Place the tube strip in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant
Slowly dispense 200 µL , or enough to cover the beads, of freshly prepared 80% ethanol into each tube. Wait 00:00:30 , then pipette off the 80% ethanol and discard
30s
Repeat the previous step
Remove residual 80% ethanol:
  • Remove the tube strip from the magnetic separation rack
  • Quick-spin the tube strip in a microcentrifuge
  • Place the tube strip back in a magnetic separation rack until beads separate fully from the solution
  • Pipette off residual 80% ethanol and discard
Remove the tube strip from the magnetic rack. Immediately add 40 µL of elution buffer to each tube and resuspend the beads
Quick-spin the tube strip in a microcentrifuge
Leave at Room temperature for 00:05:00 to elute DNA
5m
Place the tube strip in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with beads
Proceed to the next step of the protocol
Nuclease treatment
Add the following components in the order and volume listed below to a new microcentrifuge tube
  • Adjust component volumes for the number of libraries being prepared, plus 10% overage
  • If preparing 8 or more libraries, increase the overage to 25% to ensure there is enough reaction mix to dispense
  • For individual preps, add components directly to each sample from the previous step in the order and volume listed below, then skip RM3 steps (33 to 35)

Figure 3: Light Purple reagent is Nuclease buffer and needs 5 µL per library. Light Green reagent is Nuclease mix and needs 5 µL per library. This is a total of 10 µL per library, plus an additional 10% to account for pipetting error
Pipette-mix RM3
Quick-spin RM3 in a microcentrifuge to collect liquid
Add 10 µL of RM3 to each sample. Total volume should equal 50 µL
Pipette-mix each sample using wide-bore pipette tips
Quick-spin the tube strip in a microcentrifuge to collect liquid
Run the nuclease treatment thermocycler program
TimeTemperature
15 min37°C
Hold4°C

Proceed to the next step of the protocol

Note: it is necessary to remove the nucleases using either AMPure PB size selection or SMRTbell cleanup beads prior to safely storing the library or libraries
Cleanup with SMRTbell cleanup beads
15m 30s
Add 50 µL of SMRTbell cleanup beads to each nuclease treated library from the previous step and proceed to step 41 below
Using wide-bore pipette tips, pipette-mix the beads until evenly distributed
Quick-spin the tube strip in a microcentrifuge to collect all liquid
Leave at Room temperature for 00:10:00 to allow DNA to bind beads
10m
Place tube strip in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. It is recommended to save the supernatant in another tube strip in case of poor DNA recovery
Slowly dispense 200 µL , or enough to cover the beads, of freshly prepared 80% ethanol into each tube. Wait 00:00:30 , then pipette off the 80% ethanol and discard
30s
Repeat the previous step
Remove residual 80% ethanol:
  • Remove the tube strip from the magnetic separation rack
  • Quick-spin the tube strip in a microcentrifuge
  • Place the tube strip back in a magnetic separation rack until beads separate fully from the solution
  • Pipette off residual 80% ethanol and discard
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. Use wide-bore pipette tips when mixing
Quick-spin the tube strip in a microcentrifuge to collect liquid
Leave at Room temperature for 00:05:00 to elute DNA
5m
Place tube strip in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with beads
Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit. Calculate the total mass
Dilute 1 µL library with 9 µL elution buffer. Mix with a wide-bore pipette tip

Quantify the diluted library using the Qubit 1x dsDNA High Sensitivity Kit and calculate the actual concentration of the library (Qubit concentration x 10)
Further dilute the 1:10 library dilution to 250 pg/µL with Femto Pulse dilution buffer. Measure final SMRTbell library size distribution with a Femto Pulse system. This step is optional for libraries that will be pooled, but required for libraries that will be sequenced without multiplexing
Store SMRTbell libraries at 4 °C if sequencing within 1 week. Long-term storage should be at -20 °C . Minimize freeze-thaw cycles when handling SMRTbell libraries
Pool Barcoded Libraries
15m
Fill out the Download Express Microbial Multiplexing Calculator.xlsmExpress Microbial Multiplexing Calculator.xlsm80.2KB as follows:

Optionally specify sample IDs in the Sample Name column
Optionally input Barcode information for each sample
Add approximate genome size for each sample in the Expected Genome Size (bases) column
Add sample size information from the Femto Pulse QC of each sample before library preparation in the Avg Shear Size (bases) column
Add calculated actual sample concentration from the Qubit of each library (Step 54.2) to the Optional Sample Conc (ng/μL) column
If a Security Warning indicates Macros have been disabled., choose to Enable Content, then press Calculate (cell D3)
Add the desired total pool volume to cell C6. The spreadsheet will give an error if a volume lower than 100.0 is specified, but calculations will still occur
Pool the Calculated Volumes (μL) for each sample into a 1.5 mL DNA Lo-bind tube
Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit:
Dilute 1 µL library with 9 µL elution buffer. Mix with a wide-bore pipette tip
Quantify the diluted library using the Qubit 1x dsDNA High Sensitivity Kit and calculate the actual concentration of the library (Qubit concentration x 10)
For library pools with expected size of at least 10,000 bp, concentration of the pool should be between 20 ng/µL and 60 ng/µL . For library pools shorter than 10,000 bp, concentration of the pool should be between 6 ng/µL and 20 ng/µL

Under-concentrated library pools should be concentrated with a 1x SMRTbell bead cleanup, as follows:
Measure the exact volume of the library pool using a standard P200 pipette tip. Do this only once to reduce DNA shearing
Add an equal volume of SMRTbell cleanup beads to the library pool (1x v/v)
Using a wide-bore pipette tip, pipette-mix the beads until evenly distributed
Quick-spin the tube in a microcentrifuge to collect all liquid
Leave at Room temperature for 00:10:00 to allow DNA to bind beads

10m
Place tube in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. It is recommended to save the supernatant in another tube in case of poor DNA recovery
Slowly dispense 200 µL , or enough to cover the beads, of 80% ethanol into the tube. Wait 30 seconds, then pipette off the 80% ethanol and discard

Repeat the previous step
Remove residual 80% ethanol:
  • Remove tube from the magnetic separation rack
  • Quick-spin the tube in a microcentrifuge
  • Place the tube back in a magnetic separation rack until beads separate fully from the solution
  • Pipette off residual 80% ethanol and discard
Remove tube from the magnetic rack. Immediately add 20 µL of elution buffer and resuspend beads by pipetting 10 times or until evenly distributed. Use wide-bore pipette tips when mixing

Quick-spin the tube in a microcentrifuge to collect liquid
Leave at Room temperature for 00:05:00 to elute DNA

5m
Place tube in a magnetic separation rack until beads separate fully from the solution
Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube. Discard old tube with beads
Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit:
  1. Dilute 1 µL library with 9 µL elution buffer. Mix with a wide-bore pipette tip
  2. Quantify the diluted library using the Qubit 1x dsDNA High Sensitivity Kit and calculate the actual concentration of the library (Qubit concentration x 10)
After Qubit quantification of the correct- or over-concentrated library, further dilute the 1:10 library dilution to 250 pg/µL with Femto Pulse dilution buffer. Measure the final SMRTbell library size distribution using the Agilent 165kb Genomic DNA kit on Femto Pulse. See the protocol Using the Agilent 165kb Genomic DNA Kit on Femto Pulse for details

Over-concentrated library pools should be diluted to the appropriate concentration only after Femto Pulse confirms the library size
Store SMRTbell libraries at 4 °C if sequencing within 1 week. Long-term storage should be at -20 °C . Minimize freeze-thaw cycles when handling SMRTbell libraries
Proceed to Loading SMRTbell 3.0 Libraries onto the Sequel IIe when ready to begin sequencing
Protocol references