RNA-seq of SpaCas12f1
Briefly, total RNA was extracted using an EasyPure RNA Kit (TransGen Biotech Co.) from the E. coli carrying the CRISPR-SpaCas12f1 system. The total RNA was treated with 2 units of DNase I (NEB) at 37C for 30 min and subsequently purified by phenol/chloroform extraction. The DNA-free RNA was treated with 20 units of T4 PNK (NEB) at 37C for 6 h for 2-3-dephosphorylation. Then, 1 mM ATP was supplemented into the reaction and incubated at 37C for 1 h for 5-phosporylation. After phenol/chloroform extraction, the RNA was then treated with 5 units of RppH (NEB) to hydrolyze 5 pyrophosphate at 37C for 1 h. Finally, the RNA was purified by phenol/chloroform extraction. The small RNA library was constructed using the pre-treated RNA as input employing a VAHTS Small RNA Library Prep Kit for Illumina (Vazyme Biotech) according to the manufacturers instruction and then purified by 1.5VAHTS DNA Clean Beads (Vazyme Biotech). The small RNA library was subjected to Illumina HiSeq sequencing.