Mgieasy rna directional library prep kit

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.

GSC11 (3.5×10⁵ cells/well) was seeded in a 6-well plate (laminin-coated). Cells were transfected with either shNT control or shMEIS1_72 lentivirus and then incubated for 48 h. At the end of the incubation period, cells were resuspended in TRIzol® Reagent (Invitrogen; CA, USA) and kept at −80°C. Libraries were constructed using the MGIEasy RNA Directional Library Prep Kit (MGI Tech., Shenzhen, China) following the manufacturer’s procedure. RNA quality control and high-throughput RNA sequencing were performed on the MGISEQ platform (MGI Tech) with 149 bp paired-end reads. Reads were mapped to the hg19 reference genome and expression values were calculated in fragments per kb transcript per million fragments mapped (FPKM). LAS Co., Ltd. (Gimpo, South Korea) performed the RNA purification, library preparation, and sequencing procedures. The new generation of high-throughput sequencing was used to study the differences between the two samples and gene expression in both groups was compared using EdgeR. Significant genes (Log2(FC) cut-off >0.5 and <0.5; p ≤ 0.05) were used for functional annotation on the DAVID website. GSEA analysis was also performed to identify the downregulated gene sets after MEIS1 knockdown.

Total RNAs were isolated from iPSCs treated with 5 μg/mL and 10 μg/mL ECR using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and RNA quality was assessed by 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). The library was prepared using the MGIEasy RNA Directional Library Prep Kit and sequenced using MGISEQ-2000 (MGI Tech, Shenzhen, China) to generate 100-bp paired-end reads. Reads were trimmed using Trim Galore to remove adapter sequences and reads with low sequence quality. High-quality sequence reads were mapped to the human genome (hg38), and the expression levels of mRNAs were quantified using the DESeq2 [19 (link)]. The differences in expression levels between ECR treatment and control groups, in terms of the rate of change (log transformation) and statistical significance (false discovery rate; FDR < 0.01), were analyzed using the edgeR package [20 (link)] in R. ClueGO [21 (link)], a Cytoscape plug-in tool, was used to functionally grouped gene ontology (GO) and pathway annotation networks.