RNA-seq of Cas12hacker
The p15A-based plasmid encoding the CRISPR-Cas12 hacker locus was transformed into E. coli DH5 strain. Total RNA was isolated using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa). To eliminate DNA contamination, 15 g RNA was treated with 2 units of DNase I (NEB) at 37C for 30 min, followed by phenol/chloroform extraction. The RNA was then subjected to 2-3-dephosphorylation by incubating with 20 units of T4 PNK (NEB) at 37C for 6 hours. Subsequently, 1 mM ATP was added, and the mixture was incubated at 37C for 1 h to achieve 5-phosporylation. The phenol/chloroform purified RNA was treated with 5 units of RppH (NEB) at 37C for 1 h to hydrolyze the 5 pyrophosphate. Following another phenol/chloroform extraction, the pre-treated RNA was qualified by agarose gel electrophoresis and used to construct the small RNA library using VAHTS Small RNA Library Prep Kit for Illumina (Vazyme Biotech) according to the manufacturers instruction. The RNA library was purified using VAHTS DNA Clean Beads (Vazyme Biotech) and sequenced on the Illumina HiSeq platform (PE150) at HaploX Genomics Center (Jiangxi, China). The sequencing reads were mapped to the reference sequence using BWA. The mapped reads were analyzed using SAMtools. For the RNA extracted from the Cas12 hacker-sgRNA_v3 RNP complex, the similar methods for sample preparation were applied.