GSM8886959: wild-type, RNase only, rep 3; Mus musculus; RNA-Seq
Liver tissues from 8-9 days old WT and Adar1-/-/Mavs-/- mutant mice were extracted and weighed, with 100 mg used for total RNA isolation. Briefly, 1 mL of homemade TRIzol and 1.4 mm zirconium oxide beads were added to 100 mg of liver tissue in 2 mL screw cap tubes. The tissues were homogenized using a tissue homogenizer (MP Biomedicals). Following homogenization, 200 μL of chloroform was added and mixed well. Samples were centrifuged at 4°C, 15,000 rpm for 15 minutes to separate into layers. The aqueous layer was collected, and an equal volume of PCI (phenol:chloroform:isoamyl alcohol) was added, followed by another centrifugation at 4°C, 15,000 rpm for 10 minutes. The aqueous layer was again collected, and an equal volume of chloroform was added, followed by centrifugation at 4°C, 15,000 rpm for 10 minutes. To the final aqueous layer, 2x the volume of 96% ethanol and 1/10 volume of 3M sodium acetate (pH 5.3) were added. The tubes were kept at -20°C for a minimum of 2 hours or overnight for RNA precipitation. Afterwards, the samples were centrifuged at 4°C, 15,000 rpm for 15 minutes. The supernatant was discarded, and 1 mL of 70% ethanol was added to the tubes, which were centrifuged again under the same conditions. The supernatant was discarded, and the pellets were dried at 37°C for approximately 5 minutes. Finally, the pellets were resuspended in the desired volume of nuclease-free DEPC-treated ddH2O. The RNA samples were treated with DNase I (NEB) to remove genomic DNA contamination. For each sample, 10 μL of 10X DNase I Reaction Buffer, 15 μL DNase I (DNase-free), 1 μL RNase Inhibitor, and 74 μL nuclease-free ddH2O were added. The mixture was incubated at 37°C for 2 hours. After DNase I treatment, samples were extracted with 2x PCI, 1x chloroform, and 96% ethanol. RNA was quantified by Qubit fluorimetry (Thermo Fisher, Waltham, MA). Total RNA isolated from the liver tissue of ADAR1 WT and ADAR1 deficient mice was kept at 70°C for 5 minutes and allowed to refold in a buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM MgCl₂ at room temperature for 15 minutes. The RNA was then incubated with recombinant purified MDA5Δ2CARD protein in a buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM MgCl₂, and 2 mM DTT. Next, 2 μg of total RNA from WT and ADAR1 deficient mice was incubated with increasing amounts of recombinant MDA5Δ2CARD protein (100 μM-400 μM) in the presence of 1 mM ATP at 25°C for 10 minutes. The samples were then treated with a 1:100 dilution of RNase A/T1 mix (Thermo Scientific) for 5 minutes to digest unbound RNAs. Untreated samples and samples treated with only RNA and RNase served as controls. RNase A/T1 digestion was quenched by adding five volumes of TRIzol reagent, and RNA was isolated using the 2x PCI, 1x CI, and 96% ethanol precipitation method. The samples were then loaded onto an 8M Urea PAGE (6% acrylamide) gel to check for protected RNA species. Protected RNA was purified using the RNAeasy Mini Kit (QIAGEN) to remove small digestion products. rRNA was depleted with the NEBNext rRNA Depletion Kit (E6310, E6350). Using the manufacturer's protocol, cDNA libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760, E7765). Libraries from control samples (no MDA5 and no RNase) and MDA5-protected samples from both WT and ADAR1 mutant groups were diluted to 0.5 nmol. For the final library pool, 2 µl of each library was used. Sequencing was performed on an Illumina NextSeq instrument with single-ended 75 bp reads.