Clariom s array

RNA was extracted from cultured cells using Maxwell^® RSC simplyRNA Cells Kit on a Maxwell AS2000 equipment (Promega, Madison, WI, USA). Quantity and quality of RNA were verified on an Agilent 2100 Bioanalyzer. cDNA synthesis, labeling and hybridization on Clariom S Arrays from Thermo Fisher Scientific were performed by the Genomics Unit of Josep Carreras Leukaemia Research Institute. Raw data files were preprocessed using RMA (oligo R package). Gene set enrichment analysis was performed using GSEA, comparing with MSigDB hall mark gene set [77 (link)]. Full input dataset and output in HTML, specifying the running parameters, are provided as Supplementary Material.

Total RNA was extracted using TRIzol (Thermo Fisher, Waltham, USA) and the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, USA) according to the manufacturer’s instructions. Genome-wide expression analyses were performed using Clariom S arrays (Thermo Fisher Scientific) using 100 ng RNA of each sample according to the manufacturer’s recommendations (WT Plus Kit, Thermo Fisher Scientific). Data was analyzed using Transcriptome Analyses Console (Thermo Fisher Scientific, version 4.0) and expressed in log₂ values.

Clariom S arrays (Thermo Fisher Scientific) were used for global transcriptome profiling of primary adipocyte cultures treated with siRNA against ZNF436 (n = 6), NUP85 (n = 6), or respective non-targeting siRNA control (n = 6) at day 6 of differentiation. The arrays were pre-processed in Transcriptome Analysis Console (Thermo Fisher Scientific) using SST-RMA, which includes normalization, summarization of probes to probesets, and background correction. We excluded 6,341 (ZNF436 samples) and 6,316 (NUP85 samples) probe sets with detection p > 0.05 in at least one sample i.e., not detected in at least one sample, leaving 15,107 and 15,132 probe sets, respectively, for downstream analysis.
SAMR using block permutations was used to detect differentially expressed genes^{21 (link)}. Webgestalt (www.webgestalt.org) was used to identify differentially expressed gene ontologies between siRNA treated and control samples. The KEGG (https://www.genome.jp/kegg/) database was used to identify genes in the gluconeogenesis^{22 (link),23 (link)}, and wikipathways (https://www.wikipathways.org) in the fatty acid biosynthesis pathway.

RT-qPCR was performed by a two-step reaction. RNA from each cell line was prepared using RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was then synthesized using a PrimeScript RT reagent kit (Takara, Shiga, Japan), and qPCR was conducted using a TaqMan Fast Advanced Master Mix kit (Applied Biosystems, Waltham, MA, USA). Primers for RFC1 (Hs01099126), DNA methyltransferase 3β (DNMT3B; Hs00171876), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs2786624) were purchased from Applied Biosystems. The microarray analysis was performed by Clariom S Arrays (Thermo Fisher Scientific K. K, Tokyo, Japan) using 10 ng total RNA extracted from parental and PDX-resistant cells.

Six-week-old male ICR mice were treated with taurine for 8 weeks with 2% (w/v) taurine-containing drinking water. RNA from their livers was isolated using Sepazol-RNA Super G (Nacalai Tesque, Kyoto, Japan) and cleaned using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). A microarray analysis was performed on two groups (control and taurine-treated mice, n = 3 for each group) using Clariom S arrays (Affymetrix). The array was scanned by using GeneChip^TM Scanner 3000 7G (Thermo Fisher Inc., Waltham, MA, USA) and image analysis was performed by using Expression Console^TM Software (Thermo Fisher Inc., Waltham, MA, USA). These procedures were performed by Filgen Inc. (Nagoya, Japan). The microarray data are posited in the Gene Expression Omnibus.

Microarrays were performed using Clariom S Arrays (Affymetrix; Thermo Fisher Scientific) as previously described (16 (link)). Briefly, RNA was isolated using miRVANA microRNA isolation kit (Thermo Fisher), and 100 ng were hybridized onto the arrays according to the manufacturer’s protocol. Further details about data processing and analysis are given in the supplement.

Microarray experiments were performed on human Clariom S Arrays (Affymetrix) (BMFZ, Düsseldorf).