title: GSM5735492: IP_control_1; Pinus pinaster; RIP-Seq
library_construction_protocol: Total root RNA from maritime pine seedlings was isolated following the protocol described by Liao et al. (2004, Preparative Biochemistry & Biotechnology, 34: 209-214) and modified by Canales et al. (2012, Frontiers in Plant Science; 3:100). The RNA concentration and purity were determined via spectrophotometry on a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, USA). Purity was determined through the 260/280 and 260/230 ratios. RNA quality was also determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA BR, Broad-Range, Assay Kit (Invitrogen, Paisley, UK). Samples with a RIN value > 7 were selected to mRNA isolation. The poly(A)-RNA isolation was performed using Dynabeads™ mRNA Purification Kit (Invitrogen, Paisley, UK) following the manufacturer's instructions. This process was carried out twice per sample to avoid rRNA contamination. poly(A)-RNA quality was determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA HS, High Sensitivity, Assay Kit (Invitrogen, Paisley, UK). To perform the N6-methyladenosine (m6A) RNA immunoprecipitation, fragmented polyA mRNA in a range of 3.15 - 6 µg was used. Samples were precipitated with 1/10 of the total volume of sodium acetate (3M, pH5.2), 1/14 of the total volume of molecular grade glycogen (5mg/mL) (Invitrogen, Paisley, UK) and 2.5 volumes of cold molecular grade absolute ethanol. Subsequently, samples were incubated at -80 ºC overnight. After that, samples were centrifuged at 14.000 rpm for 30 minutes on a precooled ultra-centrifuge. Pellets were washed with 70% ethanol. The final pellets obtained were dried at room temperature for 10 minutes and resuspended in molecular grade distilled water (10 µL of molecular grade distilled water (Ambion, Austin, TX, USA) per 1 µg of polyA mRNA used). Samples were mixed with 10 µL of RNaseOUT (Life Technologies, Carlsbad, CA, USA), 200 µL 5X IP buffer (50 mM Tris HCl pH7.4, 750 mM NaCl, 0.5% vol/vol Igepal[CA-6300]) and 5 μL of m6A antibody (ab151230, Abcam, Cambridge, UK). The volume of the samples was brought up to 1 mL with molecular grade distilled water. Then, samples were incubated at 4ºC for 3 hours. During the time of the immunoreaction, Dynabeads™ protein-A beads (Invitrogen, Paisley, UK) were prepared using 1X IP buffer supplemented with molecular grade BSA (0.5 mg/mL) (Sigma, San Luis, MO, USA). Protein-A beads solution was rotated at 4ºC for 2 hours. Then, 100 µL of protein-A beads was added to each immunoreaction tube and incubated at 4ºC for 2 hours. Subsequently, samples were spin down, and the resulting pellets were washed three times with 1X IP buffer. For each pellet, 95 µL of proteinase K buffer (5 mM Tris pH 7.5, 1 mM EDTA, 0.25% SDS) and 5 µL of molecular grade proteinase K (Invitrogen, Paisley, UK) was added. Proteinase K reactions were incubated at 50ºC for 45 minutes and mixed every 10 minutes. Afterwards, samples were spun down, and pellets were washed twice with 300 µL of 1X IP buffer. The resulting mixtures were precipitated (as described above). Finally, pellets were resuspended in 20 µL of molecular grade distilled water. Final m6A-RNA immunoprecipitation yield was quantified using Qubit RNA HS, High Sensitivity, Assay Kit (Invitrogen, Paisley, UK). Total amount from 80 up to 130 ng were obtained. The RIP sample sequencing was carried out by Novogen (HK). The cDNA synthesis and amplification, and the library preparation was made with the SMART-Seq™ v4 Ultra™ Low Input RNA Kit for Sequencing (Takara, Mountain View, CA, USA) following the manufacturer's instructions. RNA sequencing was made in a NovaSeq 6000 sequencer according to the manufacturer's instructions for paired-end reads and final reads of 150 bp length (Illumina, San Diego, CA, USA).