T. thermophilus HB27c cells expessing dLalCas12r-CRISPR locus containing two spacers against phiKo and phiFa phages, replica 2
Small RNAs were extracted using the RNA Purification from Lira Reagent Kit (Biolabmix, Russia) according to the manufacturers microRNA purification protocol. The resulting small RNA samples were treated with RNase-free DNase I (Thermo Fisher Scientific). Libraries were prepared from enriched small RNA using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, USA) according to the manufacturers protocol. The protocol was followed up to the 12-cycles of PCR amplification, after which the reaction product was purified using magnetic beads. Quality was assessed using the Agilent High Sensitivity D1000 ScreenTape Assay on a 4200 TapeStation System (Agilent Technologies Inc., USA) and quantified with the Qubit High Sensitivity dsDNA Assay Kit (Invitrogen, Thermo Fisher Scientific, USA). PCR amplicons were proceeded to end repair, MGI-compatible adapter ligation, circularization, and DNB preparation using the MGI Easy PCR-Free Library Prep Set (MGI Tech Co., Shenzhen, China) in accordance with the manufacturer's recommendations. Barcoded libraries were pooled in equimolar ratios and sequenced on the DNBSEQ-G400 platform (MGI Tech) in 2150 bp paired-end (PE) mode.