title: GSM9149762: input_GFP_dbr1-2_rep1; Arabidopsis thaliana; RIP-Seq
library_construction_protocol: For lariat RNA-seq, total RNA of 0.5 g seedlings under normal condition and cold treatment was isolated using TRIzol (Ambion). For RIP-seq,  samples (~2 g) were fast-frozen and ground to fine powder in liquid N2. The resulting fine powder was suspended in 4 ml lysis buffer (50 mM Tris-HCl-Cl pH 8.0, 200 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.2% NP-40, 1 mM DTT, 1x protease inhibitor cocktail, 1 mM PMSF, 50 μM MG132 and 1 U/µl RNase inhibitor) at 4℃ for 30 min. Then the supernatant was passed through two layers of Miracloth. After centrifuged 3 times at 15,000 g for 10 min per time at 4℃, 100 µl of each sample was saved as input. The remaining supernatant was incubated with 50 μl of lysis buffer prewashed GFP-Trap agarose beads (Chromotek, gta-20) for 3 h at 4℃. Following IP, the beads were washed two times with lysis buffer, two times with low-salt wash buffer (200 mM LiCl, 0.1% SDS, 1% NP-40) and two times with high-salt wash buffer (500 mM NaCl, 0.1% SDS, 1% NP-40). To dissociate the RNA from the protein, the beads with buffer were added equal volume RNA elution buffer (100 mM Tris-HCl (pH 8.0), 400 mM NaCl, 2% SDS and 20 mM EDTA) and 20 mg/ml Proteinase K (Thermo, EO0491) and incubated at 65°C for 2 h with rotation. RNA was isolated with Trizol reagent. To detect more intron binding sites of these proteins, the input RNA and IPed RNA were incubated with RNase R (Epicentre, RNR07250) at 37°C for 1 h. Then, RNA was purified using the RNA Clean & Concentrator-5 (Zymo Research, R1016) following the manufacturer's instructions and eluted in 10 μl nuclease-free water. Total RNA and RIPed were firstly digested by RQ1 Dnase(Promega). RNA after RNase R digetion was purified using the RNA Clean & Concentrator-5 (Zymo Research) following the manufacturer's instructions. A Ribo-off kit (Vazyme, Nanjing, China) was used to remove rRNA from the above RNase Rtreated RNA. 50 ng of RNA for each sample was fragmented with Frag/Prime Buffer (Vazyme) at 80°C for 1 min and used as the starting material for strand-specific RNA-seq library preparation using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme).