Qauntifluor one dsdna system

Total RNA (500 ng) was used to prepare whole transcriptome sequencing libraries. Whole transcriptome RNA was enriched by depleting ribosomal RNA (rRNA), and a complete transcriptome sequencing library was generated using the MGIEasy RNA Directional Library Prep Kit (MGI) according to the manufacturer’s instructions. After rRNA depletion, the remaining RNA was fragmented into small pieces by treatment with divalent cations under elevated temperatures. Then, the cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved in RT directional buffer, followed by second-strand cDNA synthesis. An additional A base was added to the cDNA fragments and then an adapter was ligated. The products were then purified and enriched by PCR to create the final cDNA library.
The double-stranded library was quantified using the QauntiFluor ONE dsDNA System (Promega, Madison, WI, USA) and 330 ng in a total volume of 60 μl or less. The library was cyclized at 37 °C for 60 min, digested at 37 °C for 30 min, and then the circularization product was cleaned up. The library was incubated at 30 °C for 25 min with DNA nanoball (DNB) enzyme. Finally, the library was quantified using the QauntiFluor ssDNA System (Promega) and sequenced using the MGIseq system (MGI) to generate 150 bp paired-end reads.

THP-1 cells were incubated with IC₅₀ concentrations of PtNPs for 24 h. Total RNA was isolated from control or treated cells using TRI reagent (Merck, Darmstadt, Germany). In total, 500 ng of total RNA was processed for the preparation of the whole-transcriptome sequencing library. Depletion of ribosomal RNA (rRNA) was performed using an MGIEasy RNA Directional Library Prep Kit (MGI Tech, Shenzhen, China) according to the manufacturer’s instruction. The total RNAs were fragmented and copied into first-strand complementary DNA (cDNA) using reverse transcriptase and random primers. Strand specificity was achieved in the RT directional buffer, followed by second-strand cDNA synthesis. Subsequently, the cDNA fragments were ligated to sequencing adapter. The products were then purified and enriched with PCR amplification. The double-stranded library was quantified using a QauntiFluor ONE dsDNA System (Promega, Madison, WI, USA). The library was cyclized at 37 °C for 60 min, and digested at 37 °C for 30 min, followed by cleanup of the circularization product. To make a DNA nanoball (DNB), the library was incubated at 30 °C for 25 min using a DNB enzyme. Finally, the library was quantified by a QauntiFluor single-stranded (ss)DNA System (Promega, Madison, WI, USA) and sequenced on the MGIseq system (MGI Tech, Shenzhen, China) with 100-bp paired-end reads.

RNA was extracted using the RiboEx reagent (GeneAll, Seoul, South Korea). RNA was prepared for the mRNA sequencing library using the MGIEasy RNA Directional Library Prep Kit (MGI) according to the manufacturer’s instructions. The products are then purified and enhanced by PCR to form the final cDNA library. The double-stranded library is measured using the QauntiFluor ONE dsDNA System (Promega). The library is followed by the cleanup of circularization products. The QauntiFluor ssDNA System (Promega) was used to quantify the library. The constructed library was then sequenced on the MGIseq system (MGI). All datasets created and analyzed in this study are deposited in the NCBI’s Gene Expression Omnibus (GSE230271).