title: GSM4411184: Root, M-81E-salt2_IP; Sorghum bicolor; RIP-Seq
library_construction_protocol: Total RNA from roots was extracted with TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's recommended procedure. The total RNA quality and quantity were analyzed with the 2100 Bioanalyzer and the RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with a RIN number > 7.0. Poly-A mRNA was isolated from more than 200 µg total RNA with oligo-dT magnetic beads (Invitrogen). The isolated poly-A mRNA was fragmented with divalent cations at elevated temperatures to produce approximately 100-nt-long oligonucleotides. The cleaved RNA fragments were incubated for 2 h at 4 °C in a solution comprising the m6A-specific antibody (Synaptic Systems, Germany), IP buffer (50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630), and BSA (0.5 μg μl−1). The mixture was then incubated with protein-A beads and eluted with elution buffer (1× IP buffer and 6.7 mM m6A). The eluted RNA was precipitated with 75% ethanol. The eluted m6A-containing fragments (IP) and untreated input control fragments were used to construct cDNA libraries according to a strand-specific library preparation method involving dUTP. The average insert size for the paired-end libraries was approximately 100 ± 50 bp We then completed a paired-end (2 × 150 bp) sequencing analysis with the Illumina HiSeq 4000 following the manufacturer's recomm ended protocol. Two biological replicates were set for each group.
attributes.tag: GEO Accession
attributes.value: GSM4411184