Turbo dnase

Cells expressing FLAG-tagged FTO were grown to 90% confluence, washed with ice cold PBS and lysed in lysis buffer (LB) containing 150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton X-100, EDTA-free Complete Protease Inhibitor Cocktail (Roche), 1 mM DTT, RNase In (Promega). Lysates were sonicated 6 × 10 s at 7% amplitude, incubated with Turbo DNase (Fermentas) for 15 min at 37°C and cleared by centrifugation. Supernatants were subsequently pre-cleared with magnetic beads without antibodies for 1 h at 4°C. FLAG M2 Magnetic beads (Sigma) were incubated with 10 μg of yeast tRNA for 1 h at 4°C. The pre-cleared extracts were applied on the pre-blocked FLAG beads and incubated for 2 h at 4°C. Beads were washed three times with LB and the bound RNA was extracted with the TriPure reagent (Roche) according to manufacturer's instructions. The isolated RNA was treated with the Turbo DNase (Fermentas) and used as a template for cDNA synthesis by Superscript III reverse transcriptase (Invitrogen) with random hexamer priming. The cDNA was subsequently analyzed by semi-quantitative or real-time qPCR.

Cells were grown to 90% confluence, washed with ice cold PBS and lysed in lysis buffer (LB) containing 150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton X-100, EDTA-free Complete Protease Inhibitor Cocktail (Roche), 1 mM DTT, RNase In (Promega). Lysates were sonicated 6 × 10 s at 7% amplitude, incubated with Turbo DNase (Fermentas) for 15 min at 37°C and cleared by centrifugation. Supernatants were subsequently pre-cleared with magnetic beads without antibodies for 1 h at 4°C. FLAG M2 Magnetic beads (Sigma) were incubated with 10 μg of yeast tRNA for 1 h at 4°C. The pre-cleared extracts were applied on the pre-blocked FLAG beads and incubated for 2 h at 4°C. Beads were washed three times with LB and the bound RNA was extracted with the TriPure reagent (Roche) according to manufacturer's instructions. The isolated RNA was treated with the Turbo DNase (Fermentas) and used as a template for cDNA synthesis by using Superscript III reverse transcriptase (Invitrogen) and random hexamer primer mix. The cDNA was subsequently analyzed by real-time PCR.

Between 3 and 5 µg RNA per sample was used as the input for rRNA subtraction. First, equal amount of rRNA pooled oligonucleotides were added and incubated in hybridization buffer (200 mM NaCl, 100 mM Tris-HCl, pH 7.4) in a final volume of 60 µl. The samples were denatured for 2 min at 95 °C, followed by a reduction of 0.1 °C/s until the reaction reached 45 °C. A total of 3–5 µl Hybridase^TM Thermostable RNase H (Lucigen) and 7 µl 10× RNase H buffer preheated to 45 °C was added. The samples were incubated at 45 °C for 30 min. The RNA was purified using RNA Clean and Concentrator^TM-5 kit and eluted in 42 µl water. Then, 5 µl Turbo DNase buffer and 3 µl Turbo DNase (ThermoFisher Scientific) were added to each sample and incubated for 30 min at 37 °C. The RNA was purified using RNA Clean and Concentrator^TM-5 kit and eluted in 10 µl water.

Digestion and depletion of RNA and/or DNA from input samples and RNP fractions were necessary prior to MS-based proteomic analysis (Supplementary Figs. 5b and 9a–c). For SILAC LC–MS/MS experiments, RNP suspensions were normalized to 3 µg/µL protein-bound RNA in 1% LiDS TE, and 33 µL were used per 100 µL reaction containing 13.3 µL RNase Cocktail, 1× RNase digest buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, and 1 mM EDTA pH 8.0), and 1× protease inhibitors (11836153001, Roche). For the comparative LEAP-RBP experiment, 10 µL of input samples containing 2.0 µg protein/µL were used per 25 µL reaction containing 0.6 µL RNase Cocktail, 1× RNase digest buffer, and 1× protease inhibitors; 20 µL of clRNP fractions containing 0.2 µg RNA-bound protein/µL were used per 50 µL reaction containing 3.1 µL RNase Cocktail, 1× RNase digest buffer, and 1× protease inhibitors. RNase digests were incubated for 2 h at 37 °C and then precipitated with 95% methanol v/v as described above. Each input sample was suspended in TE buffer and DNA was digested using 2.5 µL Turbo DNase (Thermo, AM2238) and a final concentration of 1× Turbo DNase buffer in a 50 µL reaction (15 min, 37 °C) and precipitated with 95% methanol v/v as described above. Pellets were air-dried and submitted for proteomics analysis. For long-term storage, we recommend storing precipitates in 95% methanol at −80 °C.

Total RNA was extracted with the RNeasy Kit (Qiagen). After DNase I treatment, 3 ug of total RNA was used to synthesize the cDNA using iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). cDNA was purified with MinElute PCR Purification Kit (Qiagen).
For the ADAR EA mutant RNAseq, total RNA was extracted using RNAdvance magnetic beads (Agencourt), treated using TURBO DNase (Thermo Fisher Scientific), depleted of ribosomal RNA [50 (link)], and treated again using TURBO DNase.

Total cell RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) as per the manufacturer’s instructions. Genomic DNA was removed using TURBO DNase (Thermo Fisher Scientific). After inactivating TURBO DNase with DNase Inactivating Reagent, 1 μg DNase-free RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with random primers (Promega) as per manufacturer’s instructions. qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad). Gene-specific primer pairs used to detect mature transcripts are listed in Supplementary Table 1. Relative amount of a given target RNA under targeting versus nontargeting conditions was calculated using the formula 2^-((Ct_Target–Ct_GAPDH)_{Targeting crRNA} – (Ct_Target–Ct_GAPDH)_{NT crRNA}). No-RT and no-template controls were run alongside all RT-qPCR experiments.