Sep 12, 2022

Public workspaceTwist 96-Plex (Riptide) Library Prep 

Genes, Brain, and Behavior
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkkm85l5r/v1
  • Katarina A Cohen1,2,
  • Khai-Minh H Nguyen1,2,
  • Oksana Polesskaya1,2,
  • Abraham Palmer1,2
  • 1UC San Diego;
  • 2Palmer Lab
  • CGORD
  • Palmer Wet-Lab Protocols
Khai-Minh H Nguyen
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkkm85l5r/v1
Protocol Citation: Katarina A Cohen, Khai-Minh H Nguyen, Oksana Polesskaya, Abraham Palmer 2022. Twist 96-Plex (Riptide) Library Prep . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkm85l5r/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 02, 2022
Last Modified: September 12, 2022
Protocol Integer ID: 61801
Keywords: sample barcode, plex library prep, plex, multichannel pipette, randomization, protocol, riptide
Abstract
This protocol is designed for Twist 96-PLex Library Prep. We use the EPmotion 5075 to add sample barcodes (can also easily be done manually with a multichannel pipette). This is a continuation of the "EPMotion - Normalization and Randomization" protocol.
Guidelines
It is optional to use the EPmotion in this protocol. Visit Twist Bioscience site for updated protocols.
Materials
Please see Twist 96 Plex Protocol on Twist Bioscience Site for full list of materials needed


Equipment
  • epMotion 5075
  • 8 Channel Dispensing Tool (50uL) Catalog No. 960001044
  • Qubit Flourometer
  • Bioanalyzer
  • Nanodrop
  • Pipettes
  • Multichannel pipette (10ul) if EPmotion is not available.


Consumables
  • 1-50uL epT.I.P.S. Motion Catalog No. 0030014413
  • Qubit HS Assay
  • Tapestation D1000
  • Reagent Wells
  • Ultrapure DI Water
  • Pipette Tips: 10ul, 200ul, 1000ul

Troubleshooting
Adding Primer A with EPmotion
Review the Twist 96-Plex Protocol
Note: Protocol was created for Document Version (DOC-001284 REV 2.0)
Twist Bioscience Protocol

Fill ice pan with ice chips.
Defrost 4uL randomized/normalized sample plate from the "EPMotion - Normalization and Randomization" protocol TemperatureOn ice

Open epBlue application
Download and import Download 50% GC Content Adapters.export750% GC Content Adapters.export7 and Download Low GC Content Adapters.export7Low GC Content Adapters.export7
  • Depending on the GC content of the species you are working with, choose the protocol accordingly.
  • Use 50% GC Content Adapters if samples have GC content between 40% - 60%
  • Use Low GC Primers if samples have GC content below 40%
  • Use High GC Primers if samples have GC content above 60%

Set up the EPmotion as shown on the worktable below
Worktable for High and Low GC Content Primers - 50%
Worktable for Only Low GC Content Primers

Library Prep
Follow the Twist 96-Plex Protocol
Note: Protocol was created for Document Version (DOC-001284 REV 2.0)
  • IMPORTANT: Size selection Bead Volumes used: 50ul:20ul (Option 3) worked best for us.
  • Make sure to record which pool barcode index for each library
  • PRO TIP: We try to match the last digit of the library number (Ex. Riptide 53) with the pool barcode index used.
  • In the case of Riptide 53. We would use the pool barcode #3 for this particular library.


QC
Perform on each library and record keep all QC for pooling steps
  • Nanodrop
  • Qubit (HS Assay)
  • Tapestation (D1000)

  • Libraries should have an average fragment size between 420bp - 650bp.
  • 260/280 should be around 1.80 - 2
  • 260/230 should be around 2-2.2.
  • We have been able to get good data from libraries with relatively poor nanodrop purities.
  • Qubit concentrations can widely range. We get a range from 10ng/ul - 60ng/ul