Mgiseq system

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.

SW1783 and HT29 cells were subjected to total RNA extraction using the QIAzol Lysis Reagent, according to the manufacturer’s instructions. The concentration of isolated RNA was determined using an Agilent RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany). RNA concentration was determined using a Quant-it RiboGreen RNA Assay Kit (R11490, Thermo Fisher Scientific), and RNA quality was assessed by determining the RNA integrity number (>7) and 28S:18S ribosomal RNA ratio (>1.0) using the 2100 Bioanalyzer Instrument (Agilent Technologies). Total RNA was processed to prepare an mRNA sequencing library using the MGIEasy RNA Directional Library Prep Set (#1000006386; MGI Tech, Shenzhen, China) according to the manufacturer’s instructions. The library was quantified using the QuantiFluor ssDNA System (E3190, Promega, Fitchburg, WI, USA). The prepared DNA nanoballs were sequenced on an MGISeq system (MGI Tech) with 100 bp paired-end reads.

Total RNA (1 µg) was processed to prepare the mRNA sequencing library using the MGIEasy RNA Directional Library Prep kit (MGI Tech Co., Ltd., China) according to the manufacturer’s instructions. The library was quantified using the QauntiFluor^® ssDNA System (Promega Corporation, WI, United States). The prepared DNA nanoball was sequenced on the MGIseq system (MGI Tech Co., Ltd., China) with 100 bp paired-end reads. RNA-seq raw data quality was checked using FastQC (v0.11.9). TrimGalore (v0.6.5) was used to remove the common regions of the MGISEQ adapter sequences. The cleaned reads were mapped to the human genome assembly GRCh38 (hg38) using the STAR aligner (v2.7.3a) with default settings. Transcript abundance per gene, such as expected read count or transcripts per million, was quantified by RSEM (v1.3.3) with human gene annotation GRCh38.84. The raw sequence data (FASTQ files) and pre-processed count data were deposited in the Gene Expression Omnibus with accession number GSE182007. Differential gene expression analysis between the groups (e.g., BR treatment vs. vehicle) was conducted using the edgeR package (v3.38) in R (v3.6.3), yielding a ranked list of genes based on log₂FC and adjusted p-value.