RNA/DNA oligomers

A. Purchase and prepare nucleic acid sample

1. All DNA strands were ordered from Sangon Biotech Co. Ltd. (Shanghai, China), and RNA strands were ordered separately from Sangon BiotechCo. Ltd. (Shanghai, China) and GenePharma (Shanghai, China).
2. DNA and RNA strands were dissolved in DEPC-treated water and quantified to stock solutions with 100 mM by ultraviolet (UV)-vis absorption at 260 nm with an Agilent Cary 60 spectrophotometer (Agilent Technologies, Australia).
3. All nucleic acid samples stored at -20 °C before use.

B. The buffer and procedure of RNA/DNA oligomers annealing hybridization

1. All the RNA/DNAoligomers were preparedin annealing buffer[20 mM tris-HCl (pH 7.5), 150 mM KCl, 1 mM EDTA, and 50 mM MgCl₂].
2. The annealing carried out in S1000™ Thermal cycler (Bio-Rad, USA) and procedure is as follows: 95℃ for 4 min; gradient cooling to 25°C at a rate of 0.1°C/s .

C. Construction of cgRNA

1. 100 mM DNA and RNA was diluted to 20 mM with DEPC-treated water.

2. cgRNA was prepared as follows:

3. Annealing reaction.

a. Note: For betterperformance, we suggestthat RNA/DNA oligomers should be used as soon as possible.

D. Construction of scgRNA-1, scgRNA-2 and scgRNA

1. scgRNA-1, scgRNA-1 and scgRNA were prepared as follows:

2. Annealing reaction.

1. Note: For betterperformance, we suggestthat RNA/DNA oligomers should be used as soon as possible.