title: GSM8682995: Pellet_MKIP1_rep2; Arabidopsis thaliana; RIP-Seq
library_construction_protocol: Soluble cell components were extracted by homogenizing 10 g of seedlings per replicate in a pre-cooled blender using 30 ml of ice-cold extraction buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 1% (v/v) Triton X-100, 1 mM EDTA, 4 mM MgCl2, 1x protease inhibitor cocktail [cOmplete EDTA-free, Roche], 40 U/ml RNase inhibitor [RiboLock RNase Inhibitor, Thermo Fisher Scientific]). The homogenates were filtered through two layers of Miracloth (Milipore) and cleared by two sequential centrifugation rounds at 4,000 g for 10 minutes at 4 °C. Per replicate, 20 ml of the supernatant were incubated with 150 µl pre-equilibrated magnetic agarose anti-YFP/GFP beads (GFP-Trap, ChromoTek) for two hours at 4 °C with rotation, washed three times with ice-cold wash buffer 1 (same as extraction buffer, but replacing Triton X-100 with 0.5 % [v/v] NP-40), followed by three washes with ice-cold wash buffer 2 (50 mM Tris–HCl, pH 8.0, 150 mM NaCl). In the last wash, 10% (v/v) of the resuspended beads were collected in a separate tube and used for immunoblot analysis, while the residual beads were transferred to a new microcentrifuge tube and were processed for RNA extraction. For RNA extraction, 500 µl of input soluble extract or immunoprecipitation-processed beads were brought to a total volume of 1 ml with DEPC-treated water, mixed with 1 ml phenol:chloroform:isoamyl alcohol (Roth Lot X985.1) and subjected to phenol-chloroform RNA extraction. Two µg input RNA was treated with TURBO DNase (Thermo Fisher Scientific) in a 50 µl reaction according to the manufacturer's instructions. From this, 600 ng purified RNA was subjected to rRNA depletion treatment. The RNA was eluted in 18 µl of RNase-free water and supplemented with 2 µl RNA fragmentation buffer (400mM Tris-acetate pH 8.3, 1 M potassium acetate, 300mM magnesium acetate), followed by incubation at 94 °C for 5 minutes. Both pellet and input RNA were treated with T4 polynucleotide kinase (PNK; Thermo Fisher Scientific). Library preparation for RNA sequencing was performed using the NEXTflex small RNA-seq kit v3 (Perkin Elmer, cat# NOVA-5132-06) according to the manufacturer's instructions. The resulting cDNA was amplified with a barcode incorporated in the primer and purified according to the instructions of the NEXTflex kit.