RNA Extraction and Quantification Protocol

The yields and purity of extracted RNA were assessed by using a denovix spectrophotometer. The absorption at UV 260 nm was used to assess the RNA yields, and the ratio of 260 nm and 280 nm was used to evaluate RNA purity. An Agilent 4150 Bioanalyzer and the RNA 6000 Nano LabChip kit were employed to calculate the RNA Integrity Number (RIN). The RIN index ranges from 1 to 10, with 1 indicating the greatest degradation and 10 being the most intact RNA (Schroeder et al., 2006 (link)). The copy number of β-actin was detected on a digital droplet PCR (ddPCR) platform. Briefly, RNA in each group was reverse-transcribed into cDNA with the ReverTraAce qRT–PCR RT Kit (TOYOBO, Cat: FSQ-101). The aqueous ddPCR mixture containing 10 µl of ddPCR™ EvaGreen Supermix (BIO-RAD, Cat: #1864033), 3 µl of β-actin primers (3.75 µM) and 7 µl of cDNA was emulsified into picoliter droplets of thermostable oil in a QX200™ Droplet Generator. Subsequently, β-actin was amplified on a QX200 PCR system (Bio-Rad) at 95°C (30 s) and 60°C (60 s) for 40 PCR cycles. The ramp rate between any two consecutive steps was set to 2°C to ensure reliable thermal control. Next, the positive versus negative droplets were read by a QX200™ Droplet Reader, and the absolute quantification of β-actin was calculated using QuantaSoft software (Bio-Rad). Primer sequences for ddPCR are listed in Supplementary Table S5.