G2505c array laser scanner

HK-2 cells were stimulated with TMAO (300 µM) for 6 h at 37 °C in 5% CO₂. Total RNA was isolated from the HK-2 cells using the E.Z.N.A. Total RNA Kit I. RNA quality and integrity were evaluated using Agilent TapeStation 2200 platform (Agilent Technologies, Palo Alto, CA, USA) according to manufacturer instructions. The RNA integrity number (RIN) was 10 for all RNA samples. The Low Input Quick Amp WT Labelling Kit (Agilent) was used to prepare labelled cRNA according to manufacturer instructions. Hybridization of the labelled cRNA samples were done in a G2545A hybridization oven (Agilent) onto Agilent SurePrint G3 (v3) Human Gene Expression 8 × 60 k (Agilent Technologies) glass arrays according to manufacturer instructions and subsequently scanned with a G2505C array laser scanner (Agilent Technologies). Feature Extraction Software (version 10.7.3.1, Agilent Technologies) was used for image analysis and data extraction, as previously described [54 (link)]. Gene expression data are available in the GEO database with the accession number GSE210692.

Total RNA was isolated from the primary renal fibroblasts using the RNeasy Mini Kit (Qiagen Technologies, Hilden, Germany) according to manufacturer instructions. RNA quality and integrity was analysed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) according to manufacturer instructions. The RNA integrity number (RIN) was above 9 for all samples. Total RNA was used to prepare labelled cRNA with the Low Input Quick Amp WT Labelling Kit (Agilent) according to manufacturer instructions. Hybridization of the labelled cRNA samples were done in a G2545A hybridization oven (Agilent) onto Agilent SurePrint G3 Human Gene Expression 8 × 60 k (Agilent Technologies) glass arrays according to manufacturer instructions and subsequently scanned with a G2505C array laser scanner (Agilent Technologies). Feature Extraction Software (version 10.7.3.1, Agilent Technologies) was used for image analysis and data extraction. Gene expression data is available in the GEO database with the accession number GSE124917.

The 5637-bladder epithelial cell line (Cas9 controls and NLRP3-deficient cells) was infected with CFT073 for 6 h at a multiplicity of infection (MOI) of 10 at 37 °C with 5% CO₂. Total RNA was isolated from the cells utilizing the E.Z.N.A. Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA). RNA quality and integrity was assessed using the Agilent Tapestation 2200 platform (Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number (RIN) was 10 for all samples. The Low Input Quick Amp WT Labelling Kit (Agilent) was used to label cRNA. Hybridization of labelled cRNA samples was carried out in a G2545A hybridization oven (Agilent) onto Agilent SurePrint G3 (v3) Human Gene Expression 8 × 60 k (Agilent Technologies, Palo Alto, CA, USA) glass arrays. The arrays were then scanned with a G2505C array laser scanner (Agilent Technologies, Palo Alto, CA, USA). The feature Extraction Software (v. 10.7.3.1, Agilent Technologies, Palo Alto, CA, USA) was used for image analysis and data extraction [30 (link)]. Gene expression data is available in the GEO database with the accession number GSE243098.