E. coli RNA treated with inactive RfxCas13a (1 min, Replicate A)
Total E. coli RNA (10 g) in solution with Cas13a:crRNA complex added with either aRNA or water (negative controls) to start cleavage. Time points taken at 1, 5 and 30 min quenched with TRI-reagent. RNA clean-up conducted using the Direct-zol RNA microprep kit (Zymo) as per manufacturer's instructions. rRNA was depleted using MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen). Fragmentation carried out with RNA fragmentation reagents (Invitrogen) at 70C for 1 min followed by RNA clean-up. T4 PNK (NEB) treatment was conducted at 37 C for 1 hour before RNA clean-up. Library preparation including adapter ligation and cDNA synthesis (Vazyme Small RNA Preparation Kit) was performed before size selection (<200 nt) and Ilumina paired-end sequencing.