PacBi FC5306 Nanobind Tissue Kit Instruction Manual

PacBi FC5306 Nanobind Tissue Kit

Kit specifications

Prior to starting

Buffer CW1 and CW2 are supplied as concentrates. This kit uses CW1 with a 60% final ethanol concentration. This kit uses CW2 with a 60% final ethanol concentration. Before using, add the appropriate amount of ethanol (96– 100%) to Buffer CW1 and Buffer CW2 as indicated on the bottles.

Kit storage

RNase A and Buffer CT should be stored at 4°C upon arrival.
Nanobind disks and all other buffers should be stored at room temperature (15–30°C).

Safety precautions

Buffer BL3 and Buffer CW1 contain guanidine hydrochloride. Warning! Guanidine hydrochloride is harmful if swallowed or inhaled and causes skin and eye irritation. DO NOT mix with bleach or acidic solutions.

Product use

Nanobind tissue kits are intended for research use only.

Equipment and reagent list

Equipment/reagent

|

Manufacturer (part number)

---|---

Nanobind tissue kit

| PacBio® (102-302-100)
Magnetic tube rack|

Thermo Fisher DynaMag-2 (12321D)

Wheaton 1 mL Dounce tissue grinder with tight and loose pestles

| Fisher Scientific (06-434)
TissueRuptor II|

Qiagen (9002755)

Surgical scalpel

| Fisher Scientific (22-079-712)
ThermoMixer|

Eppendorf (5382000023)

Heat block (or water bath)

| Fisher Scientific (11-715-125DQ)
Mini-centrifuge|

Ohaus (FC5306)

Micro-centrifuge

| Eppendorf (5404000413)
Platform rocker|

Thermo Scientific (M48725Q)

Mini-tube rotator

| Fisher Scientific (05-450-127)
1.5 mL Protein LoBind microcentrifuge tubes|

Eppendorf (022431081)

2.0 mL Protein LoBind microcentrifuge tubes

| Eppendorf (022431102)
14 mL round bottom tubes|

Fisher Scientific (14-956-3B)

Wide bore 200 µL pipette tips

| USA Scientific (1011-8410)
Wide bore 1000 µL pipette tips|

Thermo Scientific (2079G)

Ethanol (96–100%)

|

Isopropanol (100%)

|

UV/Vis

| Thermo Fisher Scientific NanoDrop 2000
Fluorescent DNA quantitation|

Thermo Qubit 3.0, dsDNA BR and RNA BR Assay Kits

26g blunt end needle

| SAI Infusion (B26150)

1 mL syringe

|

Fisher Scientific (14-823-30)

For all protocols

Eppendorf Protein LoBind tubes (Eppendorf #022431081 and #022431102) are highly recommended for all extractions to reduce protein contamination from tube carryover. Protein LoBind tubes are more effective in reducing carryover contamination than DNA LoBind tubes or other tubes and will result in improved UV purity.

Introduction

Nanobind is a novel magnetic disk covered with a high density of micro and nanostructured silica that can be used for rapid extraction and purification of high-quality DNA and RNA. The high surface area and unique binding mechanism give it an extraordinary binding capacity, allowing isolation of high purity, high molecular weight (HMW) and ultra high molecular weight (UHMW) DNA in a microcentrifuge tube format. It uses a standard lyse, bind, wash, and elute procedure that is common for silica DNA extraction technologies. A single disk is used in each tube. However, unlike magnetic beads and silica spin columns which shear large DNA, Nanobind disks bind and release DNA without fragmentation, to yield DNA up to megabases in length.

Kit overview

The Nanobind tissue kit is used for the extraction of HMW (50 kb – 300+ kb) DNA from animal tissue samples. It has been used to sequence a variety of animals including mammals, fish, birds, sharks, crustaceans, and mollusks on long-range genomics platforms including PacBio Sequel®/Sequel II systems, Bionano Genomics Irys/Saphyr, and 10X Genomics Chromium. The Nanobind tissue kit can also be used to process insect, worm, and fungal samples using the appropriate aux kit. Finally, it can be used to process samples covered by the Nanobind CBB kit including cultured cells, cultured bacteria, whole blood, and nucleated blood. Depending on sample type, process time is approximately 2 h for HMW DNA and 2.5–4.5 h for UHMW DNA.

High-throughput automated extractions can also be performed.

The sample information section provides extraction and sequencing results from a variety of sample types.

Protocols are listed in the HMW (50 kb – 300+ kb) DNA extraction protocols are updated frequently so please check the PacBio Documentation page for the most up-to-date list and for the current version of the protocol.

The HMW (50 kb – 300+ kb) DNA extraction protocols are recommended for most long-read sequencing applications. This includes PacBio HiFi sequencing . For standard long-read sequencing applications, superior results will be obtained using the HMW (50 kb – 300+ kb) DNA extraction protocols. Supplementary protocols for specific tissue types are provided on the PacBio Documentation page (and are listed in the sample information section.

Workflow

Sample information

The following tables illustrate extraction data from a variety of animal species and tissue types. All samples have been validated by PacBio sequencing.

Each sample was extracted using the TissueRuptor II (TR), Dounce homogenizer (DH), and/or cryoPREP protocols (CP). These protocols differ only in the upfront homogenization approach.

For most tissues, any of the protocols may be used with similar results. If only a single protocol is listed, this does not indicate that the other protocols are incompatible.

Tissues that require modifications to the standard protocols are indicated in the tables below with asterisks (*).

See the relevant Procedures & checklists (P&C) for additional insight into experimental design, sequencing guidelines, and expected results.

Fresh frozen samples generate the best results, but tissues preserved in RNAlater and ethanol may also be used as described in the preservation methods section.

Nanobind tissue kit – Validated mammalian tissues

Sample type

| Protocols tested| Input (mg)| DNA yield (µg)| 260/280| 260/230| P&C
Human skeletal (FZ)| TR, DH, CP| 25| 8.3| 1.85| 2.04|

1

Human colon (FZ)

| TR, DH, CP| 24| 18| 1.86| 2.15| 1
Human uterine fibroid (FZ)| TR, DH, CP| 25| 10.9| 1.87| 2.19|

1

Human kidney (FZ)

| TR, DH, CP| 30| 16| 1.86| 2.20| 1
Human breast (FZ)| TR, CP| 20| 60.6| 1.88| 2.33|

1, 2

Mouse brain (FZ)

| TR| 22| 5.3| 1.85| 2.03| 3
Mouse liver (FZ)| TR, DH*| 32| 23| 1.88| 2.22|

4

Mouse spleen (FZ)

| TR*| 19| 143| 1.86| 2.30| 5
Pig brain (FZ)| DH| 30| 30| 1.84| 2.32|

3

Pig liver (FZ)

| DH| 25| 17.1| 1.90| 2.05| 4
Skunk ear punch (FZ)| TR| 33| 9.9| 1.81|

1.81

|

Vole heart (FZ)

| TR| 47| 21.3| 1.87| 2.16|
Vole kidney (RL)| TR| 38| 45| 1.87| 2.17|

Nanobind tissue kit – Validated avian sample

Sample type| Protocols tested| Input (mg)| DNA yield (µg)| 260/280| 260/230|

P&C

Zebra finch muscle (FZ)

| TR| 35| 6.6| 1.85| 1.66|

Nanobind tissue kit – validated fish samples

Sample type| Protocols tested| Input (mg)| DNA yield (µg)| 260/280| 260/230|

P&C

Teleost fin clip (FZ)

| CP| 95 (P)| 9.1| 1.87| 2.03|
Teleost liver (FZ)| CP| 25| 9.0| 1.74| 1.3|

Stickleback brain/muscle (FZ)

| CP| 50| 126| 1.84| 2.14|

Stickleback liver (RL)

| CP| 15| 8.5| 1.82| 1.84|

Antarctic icefish muscle (FZ)| TR*| 206 (P)| 10.2| 1.70|

1.65

|

9

Skate testis (FZ)

| TR, DH| 80| 112.5| 1.86| 2.32|

6

Tilapia heart (FZ)

| TR| 25| 13.1| 1.86| 2.18| 11
Tilapia fin (FZ)| TR| 27| 11.6| 1.82| 2.13|

11

Tilapia testis (FZ)

| TR*| 25| 83.7| 1.86| 2.31| 6, 11
Tilapia spleen (FZ)| TR| 25| 97.2| 1.87| 2.33|

11

Tilapia kidney (FZ)

| TR| 22| 16.1| 1.86| 2.22|

11

Nanobind tissue kit – validated mollusk samples

Sample type| Protocols tested| Input (mg)| DNA yield (µg)| 260/280| 260/230|

P&C

Black mystery snail (FZ)

| DH*| 107| 16.1| 1.79| 1.41| 7
Aplysia muscle (FR)| TR| 43| 9.1| 1.87| 2.07|

12

Nanobind tissue kit – validated crustacean samples

Sample type| Protocols tested| Input (mg)| DNA yield (µg)| 260/280| 260/230|

P&C

Dungeness crab Leg

| DH*| 100| 6.6| 1.85| 1.79| 8
Red king crab leg (FZ)| TR| 116 (P)| 112.4| 1.87| 2.28|

Snow crab leg (FZ)

| TR| 550 (P)| 15.6| 1.44| 0.92|
Tanner crab leg (FZ)| TR| 750 (P)| 3.8| 1.14|

0.63

|

Prawn

| TR| 30| 2.7| 1.81| 1.66| 9

Nanobind tissue kit – validated blood samples

Sample type| Protocols tested| Input (µL)| DNA yield (µg)| 260/280| 260/230|

P&C

Human blood

| WB| 200| 5.6| 1.90| 1.90|
Woodpecker blood| NB| 10| N/A| N/A| N/A|

10

Abbreviations

• Modifications are needed to the standard protocols and described in Procedure & checklists.
• FR Fresh tissue
• FZ Frozen tissue
• RL RNAlater preserved
• TR TissueRuptor protocol
• DH Dounce homogenizer protocol
• CP cryoPREP protocol
• WB Whole blood protocol
• NB Nucleated blood protocol
• Y Yes
• C Customer extracted and sequenced
• P Pooled extractions

1. See cryoPREP tissue homogenization Procedure & checklist
2. See Human breast Procedure & checklist
3. See Mammalian brain Procedure & checklist
4. See Mammalian liver Procedure & checklist
5. See Mammalian spleen Procedure & checklist
6. See Fish testis Procedure & checklist
7. See Black mystery snail Procedure & checklist
8. See Crab muscle Procedure & checklist
9. See Fish skeletal muscle Procedure & checklist
10. See Nucleated blood Procedure & checklist
11. See Fish tissue Procedure & checklist
12. See Aplysia Procedure & checklist

Insect samples

Insect samples are not officially supported at this time with the tissue kit. Select insects can be processed with insect aux kit. Contact us for more details.

Worm samples

Worm samples are not officially supported at this time with the tissue kit. Select worms can be processed with the worm aux kit. Contact us for more details.

Fungal samples

Fungal samples are not officially supported at this time with the tissue kit. Select fungal samples can be processed with the fungal aux kit. Contact us for more details.

DNA size

The HMW DNA extraction protocols typically yield DNA in the 50 kb – 300+ kb size range. The exact size will vary depending on sample type, the quality of the starting material, and processing parameters. For most long-read sequencing applications, superior sequencing performance will be obtained using the HMW DNA extraction protocols. This includes PacBio HiFi sequencing.

Preservation methods

High quality tissue samples are the key to obtaining high quality DNA. Fresh frozen tissues generate the highest quality DNA and are the preferred sample type. However, other preservation methods such as storage in ethanol or RNAlater can be also used. With all samples, ischemic times before preservation or freezing should be avoided to minimize tissue DNA degradation.

Frozen tissues

Tissues that are frozen without a preservation medium do not require pre- treatment before extraction.

RNAlater-preserved tissues

Tissues that are preserved in RNAlater prior to freezing or storage should have excess RNAlater solution removed.
After placing the tissue on a clean, chilled surface, wick away excess RNAlater liquid using a Kimwipe.

Ethanol-preserved tissues

Tissues that are preserved in ethanol prior to freezing or storage require pre-treatment before extraction to remove the ethanol.

1. Prepare EtOH removal buffer
   • 400 mM NaCl
   • 20 mM Tris, pH 7.5
   • 30 mM EDTA
2. Place tissue into a 50 mL conical tube containing 45 mL of EtOH removal buffer and incubate on a rocker or tube rotator for 30 min at room temperature.
3. Remove and discard EtOH removal buffer.
4. Add 45 mL of fresh EtOH removal buffer and incubate on a rocker or tube rotator for 30 min at room temperature.
5. Remove and discard EtOH removal buffer, and wick excess liquid with a Kimwipe to remove as much liquid as possible.
6. Proceed into step 1 of the appropriate extraction protocol.

Tissue disruption strategies

Choosing the appropriate disruption strategy

Most tissues may be processed with the standard TissueRuptor or Dounce homogenizer protocols. Tissues that require modifications to the standard protocols are indicated in the sample information tables and accompanied by Procedures & checklists.
In addition, we provide a Procedure & checklist describing tissue disruption using a Covaris cryoPREP instrument. We have tested alternative disruption methods including liquid nitrogen grinding and homogenization with pellet pestle. We do not recommend these homogenization methods as they do not consistently and sufficiently disrupt the tissue, leading to decreased yields, reduced purity, and diminished sequencing performance.

Homogenization tips and tricks

Dounce

It is important to use the Dounce homogenizer with the tight pestle. We do not recommend the loose pestle as this may not result in sufficient homogenization of the tissue.

When using the Dounce homogenizer, push the tissue firmly into the bottom of the Dounce chamber with each stroke. Try to keep the tissue between the tip of the pestle and the bottom of the Dounce chamber for thorough homogenization. If the tissue becomes trapped above the bulb of the pestle, pull the pestle completely out of the chamber and let the tissue settle back into the chamber by gravity.

It is not necessary to twist the pestle at the bottom of the Dounce chamber to disrupt the tissue. The compression and suction forces on the tissue that are created when moving the pestle up and down are sufficient to disrupt the tissue.

The homogenate may become foamy with some tissue types. This is normal and will not cause issues with downstream processing. If foam forms, be sure to transfer all of it in step 6.

TissueRuptor II

Use the TissueRuptor on its maximum setting. Ten seconds should be sufficient for all tissues as long as the sample was adequately minced beforehand.

cryoPREP

We have extracted and sequenced various human tissues using the Covaris cryoPREP (CP02) automated dry pulverizer for upfront disruption. Please refer to the cryoPREP tissue homogenization application note for details.

Processing tips

Magnetic rack handling procedure

To capture the Nanobind disk and enable simple processing, the microcentrifuge tubes are placed in a tube rack that is used with a magnetic base. Although DNA is bound quite robustly, proper pipetting and handling will ensure thorough washing and minimize disturbance of the bound DNA. For best results, the Nanobind disk should be  captured near the top of the tube so that fluid can be easily removed from the bottom of the tube. The following procedure is recommended.

1. Remove tube rack from magnetic base and insert tubes

2. Invert tube rack, ensuring Nanobind and liquid settle into lid

3. Place magnetic base onto inverted tube rack

4. Gently rotate the magnetic base

5. Rotate magnetic base upright, ensuring Nanobind remains captured near the top

Figure 2. Recommended procedure for capturing Nanobind disk on a tube rack and magnetic base. This procedure ensures that the Nanobind disk is captured near the top of the liquid interface, minimizing disturbance of the bound DNA and facilitating processing.

Pipetting

When removing liquid from the microcentrifuge tube, the Nanobind disk should not be disturbed. Carefully insert the pipette tip against the wall opposite the Nanobind disk and remove liquid by pipetting from the liquid surface. This will minimize the chances of accidentally pipetting bound DNA. Likewise, when adding liquid, dispense against the wall opposite the Nanobind disk.

Figure 3. Pipetting procedure for removal (left) and addition (right) of liquid during wash steps. Avoid disrupting the Nanobind disk and bound nucleic acids.

Heterogeneity and viscosity (HMW DNA Only)

The extracted HMW DNA can be highly viscous and heterogeneous. This is normal and is one of the challenges of working with HMW DNA. The heterogeneity and viscosity of the DNA eluate will vary depending on sample type, DNA size, sample input, and processing parameters. More gentle processing will yield larger DNA size but will also result in higher heterogeneity and larger amounts of highly viscous, unsolubilized “jellies.” Processing that is too gentle can dramatically reduce DNA purity and yield. To minimize the challenges of heterogeneity and viscosity, we recommend that new users err on the side of being overly aggressive. Listed below are tips for working with HMW DNA.

Following elution of the HMW DNA:

Pipette mix the extracted DNA 5–10X with a standard P200 pipette. Pipette mixing will help to loosen and coax the viscous DNA into solution. Moderate amounts of pipette mixing will not significantly impact DNA length. Pipette mixing is a standard part of our DNA elution process; we routinely use it for all long-read sequencing and optical mapping applications. For greater accuracy, the pipette mixed DNA should be left overnight at RT before quantifying the concentration0.

In some cases, the extracted DNA will be very heterogeneous and contain large amounts of unsolubilized “jellies”:

The most common reason for high sample heterogeneity and low purity is insufficient mixing during lysis. More aggressive mixing will result in samples with improved purity due to more efficient lysis and digestion. Improved sample purity will lead to improved DNA homogeneity and reduced “jellies.” Aggressive mixing during lysis will not significantly impact DNA length. Mixing may be carefully scaled back by skilled users to achieve bigger DNA. For UHMW DNA, users should follow the appropriate UHMW DNA extraction protocol.

To accurately quantify the HMW DNA:

Pipette mix the DNA 5X with a standard P200 pipette again. Perform triplicate Nanodrop readings by sampling the top, middle, and bottom of the eluate. If the concentration %CV > 30, perform an additional 5X pipette mixing using a standard P200 pipette. Let the DNA rest for at least 1 hr and repeat the Nanodrop measurements.
To accurately determine the concentration of dsDNA, we recommend making triplicate measurements using the Qubit dsDNA BR assay.

If the extracted DNA needs to be used immediately after extraction:

The extracted DNA can be sheared 5X using a 26g blunt stainless-steel needle and 1 mL syringe. The needle sheared DNA can be used immediately for library preparation. Moderate amounts of needle shearing will not significantly impact DNA length. Nearly all samples we sequence have been 5X needle sheared.

HMW (50 kb – 300+ kb) DNA extraction protocols

The following protocols are available for HMW (50–300+ kb) DNA extraction. They are recommended for most long-read sequencing applications. This includes PacBio HiFi sequencing. For standard long-read sequencing applications these protocols will result in superior sequencing performance than the UHMW (50 kb – 1+ Mb) DNA extraction protocols.

Two standard tissue protocols are provided. These protocols differ only in which upfront homogenization method was used, TissueRuptor II or Dounce homogenizer. Most tissues can be processed using either protocol.

Supplementary protocols are provided on our website.

The sample information section provides additional guidance regarding protocol selection.

HMW DNA extraction – standard TissueRuptor tissue (102-574-600)

This protocol describes the extraction of HMW DNA from animal tissue using Qiagen TissueRuptor II for sample homogenization. This protocol has been validated for many mammalian, avian, and fish tissue types including heart, liver, spleen, kidney, and colon. This protocol requires the Nanobind tissue kit (102-302-100).

HMW DNA extraction – standard Dounce homogenizer tissue (102-573-700)

This protocol describes the extraction of HMW DNA from animal tissue using a Dounce homogenizer for sample homogenization. This protocol has been validated for many mammalian, avian, and fish tissue types including gonad, kidney, liver, brain, and colon. This protocol requires the Nanobind tissue kit (102-302-100).

HMW DNA extraction – cultured cells (102-573-600)

This protocol describes the extraction of HMW DNA from cultured cells. This protocol has been validated on several cell types including GM12878 and MCF-7. This protocol requires the Nanobind CBB kit (102-301-900) or Nanobind tissue kit (102-302-100).

HMW DNA extraction – gram-negative bacteria (102-573-800)

This protocol describes the extraction of HMW DNA from Gram-negative bacteria. This protocol has been validated on several Gram-negative bacterial species including E. coli, S. sonnei, and S. enterica. This protocol requires the Nanobind CBB kit (102-301-900) or Nanobind tissue kit (102-302-100).

HMW DNA extraction – gram-positive bacteria (102-573-900)

This protocol describes the extraction of HMW DNA from Gram-positive bacteria. This protocol has been validated on several Gram-positive bacterial species including L. monocytogenes, S. aureus, and E. faecalis. This protocol requires the Nanobind CBB kit (102-301-900) or Nanobind tissue kit (102-302-100).

HMW DNA extraction – 200 µL mammalian whole blood (102-573-500)

This protocol describes the extraction of HMW DNA from 200 μL of mammalian whole blood. It has been validated using fresh and frozen whole blood. Other volumes can also be used with modification to the protocol. This protocol requires the Nanobind CBB kit (102-301-900) or Nanobind tissue kit (102-302-100).

HMW DNA extraction – nucleated blood (102-574-000)

This protocol describes the extraction of HMW DNA from 5–30 μL of nucleated blood. It has been validated on fish, bird, and lizard blood including fresh, frozen, and ethanol preserved blood samples. This protocol requires the Nanobind CBB kit (102-301-900) or Nanobind tissue kit (102-302-100).

QC procedures

Accurate quantification of HMW DNA can be challenging due to sample inhomogeneity, often leading to concentration measurements with high concentration CVs. We recommend performing replicate Nanodrop UV/Vis, replicate Qubit BR DNA assay measurements, and a single, optional Qubit BR RNA assay measurement.

See individual HMW extraction protocols for detailed guidance.

Storage of DNA

DNA can be stored in Buffer EB at 4°C for several months. Long term storage at -20°C or -80°C can be used if necessary. Avoid freeze/thaw cycles since this can degrade high molecular weight DNA.

Troubleshooting FAQ

See individual DNA extraction protocols for details.

Customer Support

Research use only. Not for use in diagnostic procedures. © 2022 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 this document. 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, and SBB are trademarks of PacBio. All other trademarks are the sole property of their respective owners.

© 2022 PacBio. All rights reserved. Research use only. Not for use in

diagnostic procedures. PN 102-587-900 REV 02 30AUG2022

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