Sorted cell lines: EPC, EPC_acLDL, and EPC_late were grown out, collected, and RNA isolated (RNeasy mini kit, Qiagen). RNA concentration and quality was measured by NanoDrop analysis. RNA was stored in RNAase free water at -80°C until analysis. RNA was hybridized to MouseRef-8 v2.0 Beadchips (25 K, Illumina, San Diego, CA). Two separate experiments were performed independently and run in duplicate at the Stanford Gene Array Core Facility. Bead level intensity values were summarized without normalization and local background correction was applied by default using Beadstudio v3.1. Microarray gene expression data were processed and analyzed using Genespring VX (Agilent, Santa Clara, CA). Clustering was performed using non-centered Pearson correlation. Data was deposited at: http://www.ncbi.nlm.gov/projects/geo (GSE53681).
Mouseref 8 v2.0 beadchips
Manufactured by Illumina
Sourced in United States
The MouseRef-8 v2.0 BeadChips are high-density oligonucleotide arrays designed for genome-wide expression profiling of mouse samples. The BeadChips contain probes targeting over 25,000 well-annotated mouse genes, allowing for comprehensive analysis of the mouse transcriptome.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Beadstudio, by Illumina (1 mentions)
Iscan bead array scanner, by Illumina (1 mentions)
Iscan control software, by Illumina (1 mentions)
Experion, by Bio-Rad (1 mentions)
Genomestudio software, by Illumina (1 mentions)
Genomestudio software package, by Illumina (1 mentions)
Mouseref 8 v2.0 expression beadchip, by Illumina (1 mentions)
Hiscan array scanner, by Illumina (1 mentions)
Totalprep rna labeling kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop apparatus, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using mouseref 8 v2.0 beadchips
1
Transcriptomics of Sorted Endothelial Progenitor Cells
2
Microarray Analysis of Peripheral Nerve Injury
3
Illumina-based Transcriptome Analysis of Mouse Samples
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Prior to gene expression experiments, total RNA integrity was confirmed using the Experion™ automated gel electrophoresis system (BioRad, Munich, Germany). cRNA sample preparation for hybridisation on the Illumina gene expression platform was performed using the TargetAmp™ Nano-g™ Biotin-aRNA Kit for the Illumina System (Epicentre/Biozym, Hess. Oldendorf, Germany) starting with 250 ng of total RNA. Samples were hybridized according to manufacturer's instructions on Mouse Ref-8 v2.0 Bead Chips (Illumina, San Diego, USA). Each chip comprises probes of 25,700 coding and non-coding RNA transcripts. Read outs of hybridisation signal intensities were performed on an iScan Bead Array scanner (Illumina, San Diego, USA), data pre-processing including spot detection, gene mapping and averaging of replicates was performed with iScan Control Software and GenomeStudio software (Illumina, San Diego). The data are accessible through Gene Expression Omnibus series [GSE67241 ].
4
Genome-wide Gene Expression Profiling of aNSCs
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Genome-wide gene expression profiling was conducted using MouseRef-8 v2.0 Expression BeadChips (Illumina, San Diego, CA), which contain ∼25,600 well-annotated RefSeq transcript features. Total RNA integrity was determined using the Experion RNA HighSens Analysis Chip (Bio-Rad, Hercules, CA) on the Experion Automated Electrophoresis System. RNA quality indicator values were >9.0 for all samples. Total RNA was labeled using TotalPrep RNA Labeling Kit (Ambion) before incubation on MouseRef-8 v2.0 BeadChips and imaging by the HiScan Array Scanner (Illumina, San Diego, CA) at the University of Chicago Genomics Facility (Chicago, IL). Four experimental replicates of aNSCs transduced with shKdm5b or control shScr lentiviral vector were simultaneously profiled on a single MouseRef-8 BeadChip. Raw data produced from HiScan imaging were outputted with the GenomeStudio software package and GEX module (Illumina).
5
Quantifying RNA Expression and Transcriptional Regulation
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Total RNA was extracted from harvested cells using RNA Microextraction kits (QIAGEN). RNA concentrations were quantified by measuring absorbance at 260 nm and 280 nm with a Nanodrop apparatus (Thermo Scientific). Analysis of the relative expression of specific genes versus that of the normalizing Rps16 housekeeping gene was performed by qRT-PCR. In short, we calculated DCt values (Ct gene À Ct Rps16), and values for normalized transcript abundance were expressed as 2( ÀDCt ). Primers used are listed in Table S4. Gene expression profiling in transfected RAW 264.7 cells was performed using Illumina MouseRef-8 v2.0 BeadChips. Results were analyzed using the twosample Bayesian t test for microarray data (Fox and Dimmic, 2006) , using p < 0.005 and FDR < 0.1 as cutoff thresholds.
Luciferase Assays 293T cells were cultured in white opaque 96-well plates with complete phenolred-free DMEM. The following day, cells were transfected with 1.8 pmol siRNA (either siYPEL5 or universal negative control). 24 hr after siRNA transfection, cells were transfected with one of the several expression plasmids, along with one of the luciferase reporter constructs; i.e., pIFNB1-LUC, pISG56-LUC or p2xNF-kB-LUC (Baril et al., 2013) . Cells were lysed 24 hr after the second transfection, and luciferase activities were determined using a dualluciferase reporter assay system (Promega).
Luciferase Assays 293T cells were cultured in white opaque 96-well plates with complete phenolred-free DMEM. The following day, cells were transfected with 1.8 pmol siRNA (either siYPEL5 or universal negative control). 24 hr after siRNA transfection, cells were transfected with one of the several expression plasmids, along with one of the luciferase reporter constructs; i.e., pIFNB1-LUC, pISG56-LUC or p2xNF-kB-LUC (Baril et al., 2013) . Cells were lysed 24 hr after the second transfection, and luciferase activities were determined using a dualluciferase reporter assay system (Promega).
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