RNAseq of the noncoding region of CRISPR-Cas12f of Acidibacillus sulfuroxidans
Total RNA was extracted using a MiniBest Universal RNA Extraction Kit (TaKaRa) according to the manufactures instruction. 15 g of RNA were treated with 2 units of DNase I (NEB) for 30 min at 37C and subsequently purified by phenol/chloroform extraction. The DNA-free RNA was then treated with 20 units of T4 PNK (NEB) for 6 hrs at 37C for 2-3-dephosphorylation. The reaction was then supplemented with 1 mM ATP and incubated at 37C for another 1 hr for 5-phosporylation. The T4-PNK-treated RNA was subsequently purified by phenol/chloroform extraction and treated with 5 units of RppH (NEB) to hydrolyze 5 pyrophosphate for 1 hr at 37C. The resulting RNA was purified by phenol/chloroform extraction. The quality was checked by agarose gel electrophoresis.  5 g of RNA were used to prepare the small RNA library using the VAHTS Small RNA Library Prep Kit (Vazyme) for Illumina according to the manufactures instruction with modifications in library amplification that two index primers containing two different 8-nt index sequences were used to substitute the universal primer and 6-nt index primer in the kit for paired-end sequencing. The resulting small RNA library was subjected to Illumina HiSeq sequencing (PE150).