Nanodrop spectrometer nd 1000 spectrophotometer

Total RNA was extracted from HS samples using a mirVana^TM miRNA isolation kit (Cat#AM1561, Ambion) following the manufacturer’s instructions, the quantity was measured using a NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab). The RNA integrity number (RIN) was tested using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified using an RNAClean XP Kit (Cat A63987, Beckman Coulter, Brea, CA, USA) and RNase-free DNase set (Cat#79254, Qiagen GmbH, Germany). The RNA of each sample that passed quality control testing was used for the construction of the library.

Transformed E. coli of BL21 cell were grown until OD₆₀₀ of 0.4–0.6, induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 2–4 h at 37°C, as done in similar studies (Alergand et al., 2006 (link); Gruber et al., 2007 (link); Wittenberg et al., 2014 (link)). The soluble fraction of cells by ultrasonication was purified by affinity chromatography (Ni²⁺-Sepharose) using 150 mM imidazole as elution buffer (Fu et al., 2008 (link)). Purity and molecular mass of the AtPDI1 proteins was confirmed using SDS–PAGE (Supplementary Figure S1). The protein concentrations were determined using a NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab).