Anti m6a rabbit polyclonal antibody

Using TRIzol (Invitrogen) and following kit recommendations, total RNA was isolated from the LECs of the included DC patients and controls, as well as SRA01/04 cells, followed by RNA quantification and purity evaluation with a NanoDrop ND-1000 spectrophotometer purchased from Thermo Fisher Scientific. This was followed by immunoprecipitation (IP) of the extracted total RNA from the NC (n = 3) and DC samples (n = 3) with an anti-m6A antibody by referring to the manufacturer’s recommendations. In brief, we placed 2 μg total RNA and m6A spike-in control mixture into a 300 μL IP buffer supplemented with 2 μg anti-m6A rabbit polyclonal antibody (Synaptic Systems, Goettingen, Germany), and let the reaction mixture rotate head-over-tail for 2 h at 4 °C. A DynabeadsTM M-280 sheep anti-rabbit immunoglobulin G (IgG) suspension (20 μL) was blocked with freshly prepared 0.5% bovine serum albumin at 4 °C for 2 h, followed by three rinses with IP buffer (300 μL) and resuspension in the total RNA-antibody mixture prepared. The RNA was then allowed to bind to the m6A-antibody beads for 2 h at 4 °C via head-over-tail rotation. After washing the beads thrice with 500 μL 1 × IP buffer and twice with 500 μL wash buffer, and incubation with 200 μL elution buffer (50 °C, 1 h), the enriched RNA was eluted and extracted using acid phenol-chloroform for ethanol precipitation.