Bulk RNA-seq Profiling of NR and HR Patients

Methanol-fixed cells were equilibrated to 4°C, centrifuged for 5 minutes at 750g, rehydrated in Wash-Resuspension Buffer (0.04% BSA MilliporeSigma; 1mM DL-Dithiothreitol Solution, Invitrogen, Waltham, Massachusetts, USA; 0.2 U/μl Protector RNase Inhibitor, Thermo Fisher Scientific; 3x SSC Buffer, Naxo Ltd.), and lysed in 700µL QIAzol solution (Qiagen, Germantown, Maryland, USA). Methanol fixation and rehydration were performed according to the protocol approved by 10X Genomics (36 (link)). Bulk RNA was extracted from pooled cells of individual patients, with miRNeasy Micro kit (Qiagen), according to the manufacturer’s instructions. The quality and concentration of purified RNA were evaluated on 2100 Bioanalyzer with the RNA 6000 Pico kit (Agilent Technologies, Santa Clara, California, USA). Samples with RNA integrity number (RIN) ≥ 7 were considered eligible for further analysis. In total, the cells from 9 NR and 9 HR patients were used for further bulk RNA-seq.