small RNA seq of processed array Fncpf1_7_spacer_array
Plasmids encoding FnCas12a and the arrays were added into 9 ul of MyTXTL master mix (Arbor Biosciences) to a final concentration of 5 nM each in a PCR tube and a total volume of 12 ul. Two uM of Chi6 annealed oligos was included in the reaction to prevent any unintentional degradation of the plasmids by RecBCD proteins. The mixture was incubated at 29C for five hours in a thermocycler. Total RNA was extracted using Direct-zol RNA MiniPrep kit following the manufacturers instructions (Zymo Research). After DNase treatment with Turbo DNase (life Technologies), and 3 dephosphorylation with T4 Polynucleotide Kinase (New England Biolabs), rRNAs were then depleted with the Ribo-Zero Bacteria Kit (Illumina) following the manufacturers instructions. RNA libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) following the manufacturers instructions.