Agilent rna 6000 kit

Total RNA was extracted from three biological replicates of vehicle and MG132-treated cells using total RNA isolation kit (Norgen Biotek). RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The integrity of the RNA sample was verified using the Agilent RNA 6000 kit and Agilent Bioanalyzer 2100 (Agilent Technologies). Samples with an RNA integrity number equal or above 8 were considered appropriate for further downstream analysis.

RNA samples were quantitated using the Qubit HS RNA kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific), following manufacturer’s instructions. The 260/280 and 260/230 ratios of absorbance values were used to assess the purity of RNA using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). A 260/280 ratio of ~ 2.0 and 260/230 ratio in the range of 2.0–2.2 was accepted as “pure” for RNA. Lower ratios may indicate the presence of protein, phenol, EDTA, carbohydrates or other contaminants that absorb at or near 260, 230 or 280 nm. The miRNA content was quantitated using a Qubit microRNA assay kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer, according to manufacturer’s recommendations. The integrity of the RNA was determined for all samples using an Agilent Bioanalyzer 2100 (Agilent Technologies, Diegem, Belgium) with an Agilent RNA 6000 kit (Agilent Technologies). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and < 7.0 considered degraded. The quality assessment of RNA samples is provided in Additional file 1: Figure S2, Additional file 2: Figure S3, Additional file 3: Figure S4.

mRNA was purified using a polyA Spin™ mRNA Isolation Kit (NEB, Ipswich, MA USA). Methylated RNA immunoprecipitation (MeRIP) was performed according to the instructions of the Magna MeRIP™ m6A Kit (Merck, Darmstadt, Germany). Briefly, 18 μg of mRNA was fragmented into approximately 100-nt oligonucleotides using a fragmentation buffer. Then the post-fragmentation size distribution was validated using an Agilent 2100 Bioanalyzer with an Agilent RNA 6000 Kit. The Magna ChIP Protein A/G Magnetic Beads were incubated for 1 h at room temperature with m6A-specific antibody in immunoprecipitation buffer. The mixture was then incubated with the MeRIP reaction mixture overnight at 4 °C. Eluted RNA was then prepared for RNA-seq libraries as described above (Additional file 1: Table S4) or for qRT-PCR analysis. Peak calling was conducted using R package exomePeak2 (v1.2.0) with default parameter settings [18 (link)], and target peaks were visualized by R package wiggleplotr (v1.14.0) (https://bioconductor.org/packages/release/bioc/html/wiggleplotr.html).