Agilent 2200 bioanalyzer

A549 cells were infected with WSN, CA04, or PR8 at an MOI of 1 or incubated with PBS (mock-infected) for 8 h. Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, USA). The purity of the RNA samples was evaluated using agarose gel electrophoresis and a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). RNA with an RNA Integrity Number (RIN) > 7, as assessed with an Agilent 2200 bioanalyzer (Agilent Technologies, USA), was screened. The rRNA was removed with a Ribo-Zero rRNA Depletion Kit (RiboBio, China), and linear RNA was removed by RNase R treatment. In addition, 1 μg of purified RNA was used for circRNA library construction. After removing reads that contained adapter sequences and low-quality reads, circRNAs were identified from the remaining reads using CIRI2 software. The remaining junction reads were aligned to human circRNAs in the circBase database (http://www.circbase.org/). Data from both the IAV- and mock-infected cells were used to identify differentially expressed circRNAs based on back splicing junction reads per million mapped reads (RPM) with a fold-change > 2 and p-value < 0.05. All raw data from these RNA-seq data were deposited in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) database with accession number PRJNA750806.

RNA from all the 48 samples was obtained using a standard TRI Reagent RNA extraction protocol. Briefly, approximately 50 mg of tissue was homogenized in 1 ml of TRI Reagent (Sigma, St. Louis, MO) by shaking using 1.4 mm silica beads, followed by the addition of 100 μl of 1-bromo-3-chloropropane (BCP) for phase separation. Afterwards, 500 μl of isopropanol were added for precipitation, which was followed by subsequent washes with 65–75% ethanol. RNA was resuspended in RNAse-free water and treated with Turbo DNAse (Ambion). Qiagen RNeasy Mini kit columns were used to clean up the samples, and RNA integrity was checked on Agilent 2200 Bioanalyzer (Agilent Technologies, USA). RNA-Seq libraries were prepared using the Illumina Truseq mRNA stranded RNA-Seq Library Prep Kit following standard protocols. Library quantity and quality were quantified using the Bioanalyzer 2100 (Agilent), and then sequenced on three lanes of an Illumina Hiseq 4000 as 75 base paired-end reads at the facilities of Edinburgh Genomics (UK). Raw reads were deposited in NCBI’s Sequence Read Archive (SRA) under BioProject accession number PRJNA552604.

To detect the collateral activity of RfxCas13d in vitro, we performed in vitro cleavage assay with 100 ng purified RfxCas13d protein, 100 ng synthesized tdTomato RNA, 100 ng crRNA, 2 μl RNase inhibitor (NEB), and 200 ng quenched fluorescent RNA reporters (6 nt poly-A/U/G/C), in 100 μl reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl₂, pH 7.6) [9 (link)]. Reactions were incubated at 37°C for 1 h and measured the fluorescence of RNA reporters with microplate reader. In Additional file 1: Fig. S8i, quenched fluorescent RNA reporters were replaced with total RNA extracted from HEK293T cells. Reactions were incubated at 37°C for 1 h and then RNA was purified by ethanol precipitation followed by quality control by Agilent 2200 Bioanalyzer.