Miniamp thermal cycler

To evaluate the quality and purity of total RNA, the NanoDrop 2000 (Thermo Fisher) was utilized, and the ratio of A₂₆₀/A₂₈₀ was 1.8–2.0, which qualified for RT-PCR and qPCR. RT was conducted using the One-Step cDNA Synthesis SuperMix (TransGen Biotech, China) and the MiniAmp Thermal Cycler (Thermo Fisher) to convert the total RNA into cDNA. The PCR reaction was performed as follows: 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 52°C–60°C for 30 s, and elongation at 72°C for 45. The PCR products were incubated at 72°C for 7 min. The primers of genes, including ACTB, GPR125, GFRA1, UCHL1, MAGEA4, CD90, SV40, VASA, RET, PLZF, and GPx3, were designed and were listed in Supplementary Table S3. The 2% agarose gels were used to separate the PCR products through electrophoresis, and PCR products with ethidium bromide could be visualized by Image Analysis System ChampGel 5,000 (Sage Creation, China).
The qPCR was conducted with cDNA as previously described using the SYBR Green Premix Pro Taq HS qPCR Kit on a CFX Connect Real-Time System (Bio-Rad, United States). The data were analyzed through the ΔΔCt method, and the reference gene was ACTB. The primers of the real-time PCR were listed in Supplementary Table S4.

Reverse transcription was performed using SuperScript III (Thermo Fisher Scientific, Cat.# 18080-051) and Oligo(dT)₂₀ primer (50 µM), following the manufacturer’s instructions. The cDNA was used immediately or stored at −20 °C until use.
Polymerase chain reaction was performed using Quick-Load Taq 2X master mix (New England Biolabs, Ipswich, MA, USA, Cat# M0271S), following the manufacturer instructions. The reactions were loaded into the MiniAmp Thermal Cycler (Thermo Fisher Scientific, Version 0.2.9, Cat# A37834). The thermocycling conditions are as follows. There was one cycle of 95 °C for 30 s, followed by 45 cycles of 95 °C for 30 s, 53.5–55 °C for 30 s (temperature varied, based on the primers, info provided in Table S1) and 68 °C for 1 min per 1 kb of expected PCR product length. A final cycle of 68 °C for 5 min finished the PCR reaction. PCR products were maintained at 4 °C before storage at −20 °C or gel electrophoresis.

Total DNA was extracted with DNAsecure Plant Kit (Tiangen Biotech, Beijing, China). The genomic DNA samples were fragmented by sonication (Covaris S220, Covaris, WBN, United States) to a size of 350 bp. DNA fragments were then end polished, A-tailed, and ligated with the full-length adapters for Illumina sequencing, followed by PCR (MiniAmp Thermal Cycler, ThermoFisher, MA, United States) enrichment using generic adapter P5 and P7 oligos. The DNA libraries were sequenced using NovaSeq PE150 (Illumina, San Diego, CA, United States) and paired-end reads in size of 150 bp were generated.