Collection Citation: Ester Kalef-Ezra, Ben Harvey, Katherine Roper, Christos Proukakis 2023. SureSelect XT HS2 DNA to prepare libraries for single-cell Whole Genome Sequencing (scWGS) after single-cell Whole Genome Amplification (scWGA). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p3qzg3e/v1
License: This is an open access collection 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 collection and it's working.
Created: October 09, 2023
Last Modified: May 31, 2024
Collection Integer ID: 89822
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research (MJFF)
Grant ID: 000430
Disclaimer
Acknowledgements and Funders:
We thank ICH.ZCR UCL Genomics Sequencing and Agilent for technical support.
This research was funded in part by Aligning Science Across Parkinson’s [Grant ID: 000430] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).
Note
ALL IMAGES CAN BE ENLARGED BY CLICKING AT THEM
Critical notes!
Please follow Good Laboratory Practices.
To prevent DNA contamination, clean all surfaces and equipment before use with DNA AWAY Surface Decontaminant, separate the lab in pre- and post-PCR areas, and use filtered sterile pipette tips.
For each protocol step that requires theremoval of tube cap strips, reseal the tubes with a fresh strip of caps.
In all master mixes to be used manually, include 5-10% excess volume in the calculations.
Abstract
We adapted the SureSelect XT HS2 DNA protocol to prepare libraries using as input material samples after single-cell Whole Genome Amplification (scWGA), instead of genomic DNA samples, for library preparation for Illumina single-cell Whole Genome Sequencing (scWGS). This protocol can be employed either manually (Section 2: Option A) or in combination with automation using the Bravo Automated Liquid Handling Platform (Section 2: Option B).
We have used this protocol for DNA products after single-cell Whole Genome Amplification (scWGA) by either PicoPLEX (Takara), ResolveDNA (BioSkryB) and droplet MDA (Samplix). We recommend to bead purify the PicoPLEX and ResolveDNA products prior library preparation. In all cases, the amplicon quantity and size should be assessed using Qubit and TapeStation or other methods prior to SureSelect XT HS2 DNA library preparation.
Bravo error/notification messages
Two error/notification messages are encountered when VWorks Bravo software is initializing:
1. If you encounter the G-axis error message shown below, select Ignore and Continue, leaving device in current state.
2. If you encounter the W-axis error message shown below, select Retry.
Materials
Commercial Reagents:
1. Equipment and consumables for DNA quality control:
DNA quantification. Here we used Qubit (Thermo Fisher Scientific):
SureSelect XT HS2 DNA Library Preparation Kit with Index Primer Pairs (Choose one of the following: Agilent G9981A (index 1-16), G9985A (index 1-96), G9985B (index 97-192), G9985C (index 193-288) or G9985D (index 289-384))
SureSelect XT HS2 DNA Library Preparation Kit with Index Primer Pairs (Choose one of the following: Agilent G9985A (index 1-96), G9985B (index 97-192), G9985C (index 193-288) or G9985D (index 289- 384))
Plasticware compatible with the selected thermal cycler. Here we used PCR 8-tube strips w/o caps 8-tube strips for qPCR/PCR, without capsVWR InternationalCatalog #732-1517
Low binding filtered tips (sterile)
Low-binding 1.5ml DNase/RNase-free (sterile). Here we used DNA LoBind Tubes,DNA LoBind® TubesEppendorfCatalog #0030108418
Powder-free gloves
DNA AWAY™ Surface Decontaminant, Surface decontaminant; 8.5 oz. (250mL)Thermo FisherCatalog #7010PK
Cleaning wipes for surface cleaning. Here we used Conti Washcloth Dry Brosch Direct PH5959
Consumables for SureSelect XT HS2 with automation:
PCR plate: PCR plates, 96-well, Armadillo™ Standard Semi-skirtedVWR InternationalCatalog #731-0649
Deep well plate for AMPure beads and master mixes: Nunc™ DeepWell™ Plates with Shared-Wall Technology, 96U, 1mL, sterileThermo FisherCatalog #260251
VersiCap Mat, 96-well, domed cap stripsThermo Fisher ScientificCatalog #AB1810
Agilent Bravo 250uL Pipette Tips, Sterile, Filtered, for 96LT headContributed by usersCatalog #19477-022
Reservoir, single cavity, polypropylene, 300 mL, 96 pyramids base geometry, 44 mm height, 25/pkAgilent TechnologiesCatalog #201244-100
Equipment:
Thermal Cycler with 96-well, 0.2 ml block. Here we used Multigene Optimax Gradient Thermal cycler with 96 well block (Labnet MB0520)
Manual version: Magnetic separator compatible with0.2 ml tubes, strips of plates. Here we used either Magnetic Separator - PCR Strip (Takara 635011) or ResolveDNA Dual Volume Strip Tube Magnet (BioSkryB PN100226)
Plate or strip tube centrifuge. Here we used SciSpin Mini microfuge, blue, 7000rpm (Sciquip SS-6050) and plate centrifuge (Starlab N2631-0008)
1.5 ml tube centrifuge. Here we used Mini-Centrifuge (Fisherbrand 16617645)
PCR cooler. Here we used PCR-Cooler, Blue, Capacity: 0.2 mL (Eppendorf 1019228)
General lab pipettes (single and multi-channel)
Nuclease-free filtered pipette tips sterile, if possible, use low binding tips
96-well plate mixer. Here we used Vortex mixer. Here we used WhirliMixer (Fisons Scientific 1993-520) and iSwix VT Digital Vortex Mixer (Appleton Woods ST6000)
Ice bucket
For automated version: Bravo NGS Option A robot (Agilent G5573A)
SMARTer-Seq™ Magnetic Separator - PCR StripTakara Bio Inc.Catalog #635011
Equipment
SciSpin MINI Microfuge BLUE, inc 2 rotors of SS-6058 & SS-6059
Safety Warnings:
Please follow the Safety Data Sheets (SDS) for all reagents for safe handling and safety hazards.
Acknowledgements and Funders:
We thank ICH.ZCR UCL Genomics Sequencing and Agilent for technical support.
This research was funded in part by Aligning Science Across Parkinson’s [Grant ID: 000430] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).
Note
ALL IMAGES CAN BE ENLARGED BY CLICKING AT THEM
Critical notes!
Please follow Good Laboratory Practices.
To prevent DNA contamination, clean all surfaces and equipment before use with DNA AWAY Surface Decontaminant, separate the lab in pre- and post-PCR areas, and use filtered sterile pipette tips.
For each protocol step that requires theremoval of tube cap strips, reseal the tubes with a fresh strip of caps.
In all master mixes to be used manually, include 5-10% excess volume in the calculations.
Manual version: SureSelect XT HS2 DNA System
https://www.agilent.com/cs/library/usermanuals/public/G9985-90000.pdf
Automated version: SureSelect XT HS2 DNA System Automated using Agilent NGS Bravo Option
A https://www.agilent.com/cs/library/usermanuals/public/G9985-90020.pdf
