GSM8879605: Hap1_ADAR1-E912A KI_dsRNA-seq_biological replicate 4; Homo sapiens; RNA-Seq
Freshly prepared buffers included NET-2 buffer (50mM Tris-Cl, pH 7.4, 150mM NaCl, 1mM MgCl2, 0.5% NP-40), NP-40 lysis buffer (50mM Tris-Cl, pH 7.4, 150mM NaCl, 5mM EDTA, 0.5% NP-40), and high salt wash buffer (50mM Tris-Cl, pH 7.4, 1M NaCl, 1mM EDTA, 1% NP-40, 0.5% DOC, 0.1% SDS). Protein G Dynabeads (Thermo Fisher Scientific) were washed and resuspended in NET-2 buffer. J2 antibody (15ug) was added to 100uL of beads and incubated for 1 h at room temperature on a rotator. Hap1 cells (1 × 107; WT, E912A KI_clone #1, ADAR1 KO_clone #1) were lysed with 300uL of NP-40 lysis buffer supplemented with RNasin® (80mU/uL, Promega) and cOmplete protease inhibitor (Roche), then incubated on ice for 5 min. Lysates were centrifuged at 17,000g for 5 min at 4°C. RNA for total RNA-seq was extracted from 50uL of lysate using Trizol reagent. For dsRNA immunoprecipitation, 250uL of lysate was diluted with 750uL of NET-2 buffer containing 12U of RQ1 RNase-free DNase (Promega) and 10mM MgCl2. 100uL of J2-Dynabeads were added and rotated for 2h at 4°C. Beads were washed twice with high salt wash buffer and twice with NET-2 buffer, and dsRNA was extracted using Trizol reagent. For total RNA-seq library preparation, RNA was extracted with the Direct-zol RNA MiniPrep (Zymo Research) from Trizol solution dissolving total cell lysate. To remove cytoplasmic rRNAs (5S, 5.8S, 18S, and 28S), the RiboMinus™ Eukaryote Kit (Thermo Fisher Scientific) was used, while mitochondrial ribosomal RNAs (12S and 16S) were retained.  For dsRNA-seq library preparation, RNA was extracted from the Trizol solution in which the bead complexes of the above dsRNA preparations were dissolved. RNA was purified using ethanol precipitation with GlycoBlue™ Coprecipitant (Thermo Fisher Scientific). The resulting blue RNA pellets were reconstituted in nuclease-free water and spiked with ERCC RNA Spike-In Mix (Thermo Fisher Scientific). Libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs) with multiplex oligos (New England Biolabs).