Northern Blotting of CRISPR-Cas10 Targets

Northern blotting was done as previously described^{5 (link)}. Briefly, overnight RN4220 cultures harbouring pTarget and pCRISPR (carrying Cas10^HD and either Card1 or dCard1) were diluted to exactly OD 0.15 before targeting was induced by the addition of 125 ng/ml of aTc. After three minutes, cells were spun down, lysed in PBS with 1 mg/ml of lysostaphin and 2mg/ml of lysozyme for 10 minutes. TRIzol (Thermo Fisher Scientific) was then added, and RNA was purified according to the manufacturer’s protocol. The RNA was separated by gel electrophoresis on a 6% acrylamide gel, and blotted onto a nylon filters (Invitrogen BrightStar Plus) with a semi-dry blotting unit (Fisher Scientific). The filters were incubated with ³²P-labelled oligonucleotides (IDT) overnight at 42 °C, and visualised on an Amersham Typhoon 5 Biomolecular Imager.