Gene 1.0 st array

After 24 h of treatment with 250 μM palmitate or vehicle control, cells were harvested by trypsinization and total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA). The quality and the concentrations of total RNA were assessed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA (100 ng) deemed to be of good quality (RNA integrity number (RIN) greater than 8) was processed according to the standard Affymetrix Whole Transcript Sense Target labeling protocol (Affymetrix, Santa Clara, CA). The fragmented biotin-labeled cDNA from three independent biological replicates was hybridized over 16 h to Affymetrix Gene 1.0 ST arrays and scanned on an Affymetrix Scanner 3000 7G using AGCC software. The resulting CEL files were analyzed for quality using Affymetrix Expression Console software and were imported into GeneSpring GX11.5 (Agilent Technologies) where the data was quantile normalized using PLIER and baseline transformed to the median of the control samples. The probe sets were further filtered to exclude the bottom 20th percentile across all samples as well as probe sets with expression levels with CV > 20 % across all replicates in a condition. The resulting entity list was subjected to a t-test with Benjamini-Hochberg FDR correction. The data files have been deposited in Array Express, Accession number: E-MTAB-2601.

Affymetrix Human Gene 1.0 ST Arrays were processed according to the Affymetrix Expression Analysis Whole Transcript (WT) Sense Target Labeling Protocol (Affymetrix, Inc.). Briefly, total RNA (300 ng) was converted to double-strand cDNA. cRNA was obtained by an in vitro transcription reaction and used as the template for generating a new first-strand cDNA. The cDNA was fragmented, end-labeled with biotin and hybridized to the array for 16 h at 45 °C using the GeneChip Hybridization Oven 640 (Affymetrix, Inc.). Washing and staining with streptavidin–phycoerythrin was performed using the GeneChip Fluidics Station 450 and the images acquired using the Affymetrix Scanner 3000 7G Plus (Affymetrix, Inc.). The data were normalized using quantile normalization with the RMA algorithm for gene-level intensities and the ratio determined for each gene using Partek Genomics Suite (Partek Inc., St. Louis, MO, USA). Total RNA was processed at the microarray facility from the center for applied genomics at Public Health Research Institute in New Jersey, using the Affymetrix Gene 1.0 ST Array.

Five public microarray datasets (GSE15471, GSE28735, GSE62165, GSE62452, and GSE40098) were retrieved from the publically available database GEO. LncRNA expression profiling of GEO datasets was analyzed using the Affymetrix Human Genome U133 Plus 2.0 Array platform, Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) and Agilent‐014850 Whole Human Genome Microarray 4 × 44K G4112F. The recent release of the HG‐U133Plus2 array includes 6492 probe sets corresponding to 5563 lncRNAs. R software and packages were used to preprocess RNA sequencing and microarray data.