title: GSM6671029: Arabidopsis mRNA rep2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: For acRIP-seq, seedlings were homogenized in liquid nitrogen, and RNA was harvested using Trizol reagent. ac4C IVT was spiked-in in a 1:5000 mass ratio. RNA was fragmented and isolated with IgG or ac4C antibody. For ac4C-seq, mRNA was purified by oligodT beads. 3 ug of mRNA was used for the construction of sequencing libraries. For RNC-seq, Arabidopsis seedlings were homogenized in liquid nitrogen and lysed in in 1 ml pre-chilled ribosome buffer (20 mM HEPES[pH 7.44], 15 mM MgCl2, 200 mM KCl, 100 mg/ml cycloheximide, 2 mM dithiothreitol) with 1% Triton X-100 for 30 minutes at 4ºC. Cell lysate was centrifuged at 17,000 g for 5 minutes and supernatant was taken. The supernatants were transferred to 12.5 ml sucrose buffer (30% sucrose in ribosome buffer). After centrifuging at 185,000g for 5 hours at 4ºC, the pellet was suspended in cold ribosome buffer to get RNC. RNA libraries were prepared for sequencing using standard Illumina protocols. acRIP-seq libraries were prepared according to Arango, D. et al., Cell, 2018. Briefly, RNA was converted to final cDNA libraries in accordance with a dUTP dependent strand-specific library preparation. Libraries were sequenced on the Illumina Hiseq 4000 platform as single-end 150 bp reads.   ac4C-seq libraries were prepared using NEBNext UltraTM Ⅱ Directional RNA Library Prep Kit for Illumina (NEB, E7760S) following manufacturer's protocols.