Beadarray 500gx reader

EDTA anticoagulated venous blood samples were collected from all participants. Genomic DNA was extracted from blood buffy coats using standard phenol-chloroform procedures. DNA samples were genotyped according to the manufacturer’s instructions on Illumina Infinium High Density (HD) Human610-Quad DNA analysis BeadChip version 1. BeadChips were a four-sample format requiring 200 ng of DNA per sample. Samples were scanned on the Illumina BeadArray 500GX Reader. Raw data was obtained using Illumina BeadScan image data acquisition software (version 2.3.0.13). Raw data from Illumina idat files were SNP genotyped in R using the CRLMM package [59 (link)]. Genotype data then underwent initial quality control routines using PLINK [60 (link)]. SNPs were filtered based on: minor allele frequency >0.01; call rate >0.95, and Hardy-Weinberg equilibrium testing p-value >10⁻⁵. After this initial quality control, 590,603 SNPs were exported from PLINK and imported into the CRAN package GenABEL [58 (link)]. Further filtering (including Mendelian inheritance violations and sex-checking based on available X and Y markers) in GenABEL lead to the reduction of the SNP set to a total of ~480,000; this included removal of both X and Y chromosome SNPs after gender checking, as well as the removal of mitochondrial and XY SNPs.

The expression data of RA were downloaded from Gene Expression Omnibus (GEO) DataStets (https://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS3794) and corresponding reference [21 (link)]. In the study, a complete genome-wide transcript profiling of peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 15 age- and sex-matched healthy controls were collected. The information about the demographical and clinical characteristics of RA patients and controls was presented in Table S1. The total RNA was isolated from PBMCs using the PAXgene RNA isolation kit (PreAnalytix). The concentration and profiles of total RNA were determined using a NanoDrop ND-1000 (Wilmington) and a Bioanalyzer 2100 (Agilent). Two hundred nanograms of RNA was reverse transcribed. The cDNA was transcribed and cRNA was labeled with biotin-16-UTP. Labeled probe hybridization to Illumina BeadChips human-6v2 was carried out using Illumina’s BeadChip 6v2 protocol. Beadchips were scanned on the Illumina BeadArray 500GX Reader. The preliminary data analysis were performed and normalized by Illumina BeadStudio software. Then the real-time PCR was further performed to detect mRNA expression. Differential analysis per gene was performed with one-way analysis of variance (ANOVA) and P values were adjusted to control the false discovery rate (FDR, 5%).

DNA samples were genotyped using on the Illumina Bead Array 500GX Reader. using Illumina Infinium High Density (HD) Human610‐Quad DNA Bead Chips version 1 as described previously (Cox et al. 2012). A total of 620,901 genome‐wide markers were genotyped and markers had a median spacing of 2.7 kb (mean = 4.7 kb) throughout the genome. In this study all genotyped SNPs were directly observed via lab‐based assays, that is, not imputed. Individuals with a call rate below 95% and SNPs with a call rate below 99%, deviating from Hardy–Weinberg equilibrium (p_HWE<1 × 10⁻⁷) or with a minor allele frequency of <1% were excluded.

Total RNA was extracted from 8348SOCS3 cells and 8348plv cells using Trizol reagent. Genome expression analysis was performed using an Illumina Human HT-12 v4 BeadChip (Illumina, San Diego, CA, USA) at the Beijing Qian Zhao Xing Ye Biological Technology Co., Ltd. (Beijing, China). The beadchips were scanned on the Illumina Bead Array 500GX Reader using Illumina BeadScan image data acquisition software. Illumina BeadStudio software was used for preliminary data analysis. The preliminary data were normalized using sample averages; the sample intensities were scaled by a factor equal to the ratio of average intensity of a sample to the average intensity of the given sample [8 (link)]. 8348SOCS3 cells and 8348plv cells were regarded as the given sample. Each sample was repeated three times. An Illumina custom algorithm was used to compare 8348SOCS3 cells with 8348plv cells. A difference score for a probe (diff score) indicates differential gene expression between the two groups. For each gene, the diff scores of corresponding probes were averaged. The results were validated using qRT-PCR.

Targets were synthesized, amplified, labeled, and purified using the TargetAmp Nano-G Bioti-aRNA Labeling kit (Epicentre, Madison, WI) according to the manufacturer's instructions. Briefly, 500 ng of total RNA were reversed transcribed and subject to second strand cDNA synthesis, followed by in vitro transcription and complementary RNA labeling with biotin-dUTP. Labeled targets were then subjected to hybridization to Illumina BeadChips Human HT12 according to the protocol recommended by Illumina (Illumina, San Diego, CA, USA). The chips were scanned on the Illumina BeadArray 500GX reader and images were processed by Illumina BeadScan software. Genome Studio (Illumina) and Partek Genomics Suit (Partek, St. Louis, MI, USA) software packages were used for preliminary data analysis. A model-based background correction method was used to correct the background noise and to normalize the data before analysis to obtain differentially expressed genes in testing samples [71 (link)]. Microarray data are available in the Gene Expression Omnibus (GEO) under Accession No GSE62326. Genes with less than raw signal <100 were filtered out, leaving genes for further analysis.

Total RNA was extracted from hematopoietic cells transfected with shSOCS3 or empty vector (5×10⁶ cells) using Trizol reagent (Invitrogen Life Technologies, Paisley, UK). Genome expression analysis was performed by Illumina Human HT-12 v4 BeadChip (Illumina, San Diego, CA, USA) at the Beijing Qian zhao xing ye Biological Technology Co., Ltd. (Beijing,China).
HumanHT-12 v4 Expression BeadChip targets more than 48000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (7 November 2009) and other sources. The beadchips were scanned on the Illumina Bead Array 500GX Reader using the Illumina BeadScan image data acquisition software. Illumina BeadStudio software was used for preliminary data analysis. The preliminary data were normalized using sample averages. To do so, the sample intensities were scaled by a factor equal to the ratio of average intensity of a sample to the average intensity of the given sample. In this article hematopoietic cells transfected with empty vector were regarded as the given sample. Each sample was repeated 3 times.
An Illumina custom algorithm was used to compare shSOCS3-1 CD34 ⁺ cells with control cells. A difference score for a probe (diff score) indicates differential gene expression between these two groups. For each gene, the diff scores of corresponding probes are averaged.