R-loop nick initiation protocols #

sgRNA synthesis using NEB EnGen Kit (E3322S) #

  1. Follow NEB EnGen Kit protocol using desired oligo. Detailed protocol can be found at this link and is shown in the image below.

Preform phenol-chloroform purification of RNA products (adapted from NEB E2040).

  1. Increase sample volume to 200 ul using nuclease-free water. Transfer sample to a phase-lock column. These can be commercial or homemade columns.
  2. Add 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), when pipetting make sure to use a reach tip to penetrate through the top layer of solution in the bottle.
  3. Votex sample vigorously to mix the phases.
  4. Centrifuge at 12,000 g for 5 minutes.
  5. Carefully pipette the top layer into a new tube.
  6. Add 2.2 volumes of 100% EtOH and 0.1 volumes of 3M sodium acetate, pH 5.2 and 2 ul molecular grade glycogen.
  7. Incubate at -20°C for at least 30 minutes.
  8. Centrifuge samples at 14,000 rpm for 30 minutes.
  9. Wash samples twice with 200 ul 70% EtOH.
  10. Re-suspend samples in suitable volume of 0.1 mM EDTA and store at -20C.

In vitro digestion of DNA with EnGen Spy Cas9 Nickase #

Follow NEB provided protocol, available at this link.

Validation of targeted nicks using Sanger sequencing #