T. H. (Henry) Hung [email protected], University of Oxford

📬 For any intended reader only

Graphical Abstract

Reagents for Day 1

• [ ] 0.5 Rx NEBNext dsDNA Fragmentase & Reaction Buffer (#M0348
• [ ] 2.5 µl 0.5 M EDTA (pH 8.0)
• [ ] ~80 µl AMPure XP (#A63880)
• [ ] 1.8 ml freshly-prepared 80% ethanol
• [ ] 3 Rx Qubit dsDNA BR
• [ ] 1 Rx NEBNext Ultra II DNA Library Prep Kit for Illumina (#E7645, or alternatively but more expensive:
  • [ ] 1 Rx NEBNext Ultra II End Repair/dA-Tailing Module (#E7546)
  • [ ] 1 Rx NEBNext Ultra II Ligation Module (#E7595
• [ ] 1 Rx Q5 Hot Start High-Fidelity 2X Master Mix (#M0494)
• [ ] 1 Rx NEBNext Multiplex Oligos for Illumina (Selection Chart, recommended: #E6609 for single indexing; #E7600) or #E7780 for dual indexing)

<aside> ⌛ Aliquot the amount of AMPure XP beads needed for each experiment (after vortexing to resuspend) and let it sit in room temperature for 30 min.

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<aside> ⚠️ A multichannel pipette must be used when dealing with 96-well plates to make sure addition or removal of reagents are rapid enough to maintain consistency across the samples.

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Day 1

Second-strand cDNA synthesis

🗓️ Day 1 ⌛ 80 min