Miniaturization of Nextera XT Library Preparation for RNA-sequencing of Murine Neuronal Cell cDNA Samples using the MANTIS® Liquid Handler

Introduction:

In providing various next generation sequencing (NGS) services relating to a wide range of customers, sample types, and sequencing approaches, core laboratories must balance the efficiency of their NGS workflow with the quality of the data that they generate.  As the cost of sequencing declines, the expenses associated with library preparation begin to dominate the cost of the entire NGS workflow.  In order to address this dominant expense in sequencing, many institutions are adopting the trend of reaction miniaturization, effectively reducing volumes to as little as 10% of the manufacturer recommended volume.

This application note highlights a workflow for generating high-quality NGS libraries, even from low cellular input, through precise positive displacement microfluidic reagent dispensing.  The quality control data for quarter volume Illumina Nextera XT library preparation showcase the applicability and efficacy of reaction miniaturization in the context of single-cell sequencing.

Materials:

  • Eppendorf twin.tec 384 plates (PN 0030129342)
  • Illumina Nextera XT DNA Library Preparation Kit (PN FC-131-1096)
  • Illumina Nextera XT Index Kit V2 (FC-131-2001)
  • Thermal Cycler
  • FORMULATRIX® MANTIS® Liquid Handler (PN MANTV3.2)
  • Alpaqua 384 Post Magnet Plate (PN A001222)
  • ThermoFisher Qubit 4 Fluorometer (PN Q33226)
  • Agilent 2100 Bioanalyzer (PN G2939BA)

Methods:

Quarter Volume Illumina Nextera XT Library Preparation

Eight murine neuronal cell cDNA samples (3 cell types; 1 ng DNA as input) were used in this pilot project to assess the quality of libraries produced at quarter volumes via the following protocol:

A. Tagment cDNA – 6.35 µL total reaction volume

  1. Begin with 1.25 µL of input DNA in wells of a 384-well microplate
  2. Aspirate TD buffer into a 200 µL pipette tip* and place on LV chip on MANTIS. Dispense 2.5 µL of TD buffer into each sample containing well with MANTIS.
  3. Mix
  4. Aspirate ATM into a 200 µL pipette tip* and place on LV chip on MANTIS. Dispense 1.3 µL of ATM into each sample containing well with MANTIS.
  5. Mix
  6. Thermal Cycle:
    1. 55C for 5 min
    2. 10C Hold
  7. Aspirate NT Buffer into a 200 µL pipette tip* and place on LV chip on MANTIS. Dispense 1.3 µL of NT Buffer into each sample containing well with MANTIS.
  8. Incubate at room temperature for 5 minutes

B. Amplify Libraries – 12.65 µL total reaction volume

  1. Aspirate NPM into a 200 µL pipette tip* and place on LV chip on MANTIS. Dispense 3.8 µL into each sample containing well with MANTIS.
  2. Manually pipette 1.25 µL of two index primers to samples so that each sample receives a unique combination of index primers.
  3. Thermal Cycle:
    1. 72C for 3 min
    2. 95C for 30 sec
  • 12 cycles of
    1. 95C for 10 sec
    2. 55C for 30 sec
    3. 72C for 30 sec
  1. 72C for 5 min
  2. 10C Hold

 

C. Clean-up Libraries

  1. Aspirate AMpure XP beads into 200 µL pipette tip* and place on HV chip on MANTIS. Dispense 10 µL into each sample containing well with MANTIS.
  2. Incubate at room temperature for 5 minutes
  3. Place plate on magnet and wait for 2 minutes
  4. Manually remove all supernatant (do not remove plate from magnet until step 11.).
  5. Using continuous flow on MANTIS, add 50 µL of 80% EtOH to each sample containing well.
  6. Incubate for 30 seconds.
  7. Manually remove all supernatant while plate sits on magnet.
  8. Repeat steps 5-7.
  9. Air dry for 15 minutes.
  10. Remove plate from magnet.
  11. Aspirate RSB into a 200 µL pipette tip* and place on HV chip on MANTIS.
  12. Dispense 15 µL into each sample containing well with MANTIS.
  13. Incubate at room temperature for 2 minutes.
  14. Place plate on magnet.
  15. Incubate for 2 minutes.
  16. Manually transfer 12.5 µL from beads to new plate.
*Aspiration volume = dispense volume per sample x number of samples + 10% safety factor.  For aspiration volumes above 200 µL, use a 1000 µL pipette tip.

Results:

The quality of Nextera Libraries for each sample was assessed via tracings from the Agilent BioAnalyzer.

Figure 1. BioAnalyzer tracings of eight next generation sequencing libraries prepared from 8 unique samples of 1ng murine neuronal cell cDNA

Conclusion:

The BioAnalyzer tracings indicated that the composition of libraries prepared through quarter volume reaction miniaturization was acceptable and comparable to those prepared when following the full-volume protocol.

Preparing libraries at quarter volumes results in a significant cost savings of 75% of the kit cost per reaction.  The MANTIS® Liquid Handler from FORMULATRIX® facilitates reaction miniaturization through precise microfluidic reagent dispensing at volumes as low as 100 nL and is consumable-free, simple to use, and reliable.

Acknowledgements:

This work was carried out by Xi Chen of the Comprehensive Cancer Center at The Ohio State University in collaboration with FORMULATRIX®.

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