Ferroptosis-related DNA methylation data and clinical information of HCC patients were downloaded from the TCGA database (19 (link)) (https://cancergenome.nih.gov/ ). Besides, we also downloaded gene expression data to identify the potential underlying biological mechanisms. The RNA-seq data were generated from Affymetrix Human Genome U133A 2.0 (GPL3921). A total of 375 HCC patients were included in the present study. Only patients with survival information were included in this study. We obtained 60 ferroptosis-related genes from the previous literature (17 (link)) (Table S1 ). A total of 1,035 ferroptosis-related DNA methylation sites were annotated. An additional validation set (GSE14520) was downloaded from the GEO database (20 (link)) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14520 ), which only included clinical information and RNA-seq data of 221 HCC patients. The RNA-seq data were generated from Affymetrix GeneChip HG-U133A 2.0 arrays (GPL571 and GPL3921 platforms). The clinical information including study dates, eligibility criteria, et al were in line with the published literatures (19 (link),20 (link)). The sample size was estimated using nQuery Advisor 6.01 (Statistical Solutions Ltd., MA, USA) software. A sample size of 300 participants could reach 95% power of study (two-sided α=0.05).
Human genome u133a 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human Genome U133A 2.0 Array is a microarray platform designed for comprehensive gene expression profiling. It contains over 54,000 probe sets representing approximately 47,000 transcripts and variants, which cover a large portion of the human genome.
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Lab products found in correlation
Genechip hg u133a 2.0 arrays, by Thermo Fisher Scientific (1 mentions)
Expression console software, by Thermo Fisher Scientific (1 mentions)
Human genome u133a, by Thermo Fisher Scientific (1 mentions)
Human genome u95av2, by Thermo Fisher Scientific (1 mentions)
Human genome u133a plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Ht human genome u133 array, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using human genome u133a 2
1
Ferroptosis-related DNA Methylation Profiling in HCC
2
Affymetrix GeneChip Expression Analysis
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Gene expression data were obtained using Affymetrix Human Genome U133A 2.0 or Plus2 GeneChips; mRNA isolation, labeling, hybridization, and quality control were carried out as described before (Alvarez et al., 2011 (link)). Raw data were processed using the Robust Multi-Averaging (RMA) algorithm and Affymetrix Expression Console software. Data are available in the NCBI Gene Expression Omnibus database (GSE101082).
3
Oral Squamous Cell Carcinoma Microarray Analysis
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The data were downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/ ) and were selected based on the following criteria to ensure the reliability of the data analyses: (1) availability of raw microarray data; (2) inclusion of both oral squamous cell carcinoma and normal control (either adjacent normal or oral mucosa from healthy individuals); and (3) more than 10 tumor samples. Consequently, there were 6 datasets [11 (link),12 (link),13 (link),14 (link),15 ,16 (link)] using Affymetrix microarray that met our requirements (S1 Table ). A total of 481 samples (326 OSCC and 165 normal controls) were included in this analysis. To ensure abundant availability of information for subsequent analyses, GSE9844/GSE30784/GSE31056 were labeled as “group I” (which was from Affymetrix Human Genome U133A Plus 2.0 array) and the remaining datasets were labeled as “group II” (which consisted of the former version of Affymetrix such as Human Genome U133A, Human Genome U95Av2 and Human Genome U133A2.0) according to their microarray platform.
4
Survival Analysis of Normalized Gene Expression Data
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The survival analysis is performed on ´GSE14520` provided by NCBI-GEO [19 (link)]. Therefore, the raw data is RMA-normalized per array-platform (Affymetrix Human Genome U133A 2.0, Affymetrix HT Human Genome U133 Array). Due to the same feature annotation on both platforms the annotation of hgu133a2 is used. Gene-centered information was obtained by summarizing and averaging the expressions of all gene-specific spots per array as described by the annotation of hgu133a2. After that, quantile normalizations over both platforms are applied due to the differences between both datasets. The expression values of this dataset were reduced to the cut-set of genes between the clusters identified in the gene expression analysis of ‘GSE2109’. The remaining expression matrix was centered using the mean of the respective gene and normalized via the standard deviation. Next, the expression matrix was centered using the mean of the respective array and normalized via the standard deviation. Then, arrays were grouped using hierarchical clustering. Expression values of each gene are depicted as density and boxplot, differences are described via t-test, wilcox test, fold change and log fold change.
5
Microarray Data Analysis for Chronic Kidney Disease
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The microarray data GSE48944 for CKD was downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (GEO) on May 7, 2018 (7 (link)). The testing platform was Affymetrix Human Genome U133A 2.0 (GPL571: HG-U133A_2). GSE48944 included 13 human CKD samples and 12 control (CTRL) samples. Microarray raw data (.CEL files) of the CKD were processed with oligo Version 1.41.1 (http://www.bioconductor.org/packages/release/bioc/html/oligo.html ) in R language to achieve an approximate normal distribution (8 (link)). Subsequently, the data were standardized using the median normalization and quantile methods.
6
Isoborneol Treatment Alters MCF7 Transcriptome
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After pre-treatment with 10 μM isoborneol, MCF7 cells were harvested, and total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, US) according to the manufacturer’s instructions. DMSO-treated cells were selected as a control. The gene expression profiles were assessed using microarray technology with Affymetrix Human Genome U133A 2.0 (Santa Clara, CA, US).
7
Herbal Compounds Gene Expression Profiling
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Gene expression data of herbal compounds were obtained from a systematic study on TCM components by using the gene expression microarray technique (Lv et al., 2017 (link)). These small compounds were common in Chinese herbs and TCM formulae, and most of them are the quality control components of TCMs from the China Pharmacopoeia. The gene expression data were derived from the human breast cancer epithelial cell line (MCF7) treated with these herbal compounds, profiled using microarray technology with Affymetrix Human Genome U133A 2.0, and collected from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO series accession number: GSE85871). The raw data (CEL files) were processed consistently by applying the Affymetrix platform-specific procedure to filter and normalize data sets. Then, for each herbal compound, the differential gene expression values versus control samples were calculated by R package “Limma” (version 3.32.7). The gene list for each compound was produced by ordering genes in a ranked list according to their differential expression values.
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