| GSM7567517 channel 1 extraction |
Mouse brain cortex samples or cell pellets were homogenized in 1mL TRIzol (Invitrogen) and incubated at room temperature for 5min. 200µL Chloroform was added in sample’s tube, thoroughly mixed, and incubated at room temperature for 15min. Next, all samples were centrifuged at 13,000g for 20min at 4ºC. The total RNA was precipitated in 60µL NaAc (3M, pH 5.2), 4µL glycogen (5 mg/mL), and 600µL 100% Isopropanol for at least 30min at -80ºC with 600µL supernatant. 4µL E-PAP and 40U RNase inhibitor were added to 10µg total RNA in a 50µL reaction by following the poly(A) tailing kit (Thermo Fisher Scientific). The reactions were incubated for 1hour at 37ºC, purified and eluted with 25µL nuclease-free water. For the RNase R treatment, the RNA products were treated with 10U RNase R, 3µL 10X RNase R Buffer with specific LiCl (0.2M Tris–HCl pH 8.0, 1mM MgCl2, and 1M LiCl), and 80U RiboLock RNase Inhibitor (40 U/μL, Thermo Fisher Scientific). Finally, the RNA products were purified and eluted in 30µL nuclease-free water to proceed the rRNA-depleted RNA-seq library. We used the Qubit RNA High Sensitivity Assay to quantify the A-tailing RNA y ) and the Bioanalyzer 2100 Eukaryote Total RNA Pico to value the quality of the A-tailing RNA. The Ribo-Zero rRNA Removal Kit was used to deplete ribosomal RNAs and the NEBNext UltraTM RNA Library Prep Kit was used to prepare libraries. |
1 |