GSM6965746: treatment group overexpressed both SAMD4B and APOA2, replicate1,NM-seq; Homo sapiens; OTHER
RNA was harvested using Trizol reagent. Nm-Seq service was provided by CloudSeq Biotech Inc.(Shanghai, China) by following the published procedures with slight modification (1). Briefly, the RNA samples were fragmented at 95 °C for 5 min with RNA Fragmentation Reagents (GenSeq Inc.). RNA fragments were 3′-end repaired using antarctic phosphatase (New England BioLabs) at 37 °C for 30 min. Then, repaired RNA samples were oxidized/eliminated using 10 mM NaIO4 (Sigma-Aldrich) in 200 mM lysine–HCl buffer (pH 8.5, Sigma-Aldrich) in a total volume of 40 µl at 37 °C for 30 min. The reaction was quenched by ethylene glycol, samples were further dephosphorylated by Shrimp Alkaline Phosphatase (New England BioLabs) at 37 °C for 30 min. Eight cycles of oxidation–elimination–dephosphorylation were performed. A final round of oxidation/elimination reaction was performed, excluding dephosphorylation. Then, purified RNA samples were 5′ phosphorylated by T4 polynucleotide kinase 3′ phosphatase minus (New England BioLabs) at 37 °C for 60 min. Libraries were constructed using GenSeq® Small RNA Library Prep Kit (GenSeq Inc.) and quantified using Qubit fluorometric assay (ThermoFisher).