Whole transcript sense target labeling protocol

For each sample, 300 ng of total RNA were amplified and labeled using the Affymetrix Whole-transcript (WT) Sense target Labeling Protocol without rRNA reduction (www.affymetrix.com). Ten µg of cRNA was carried out into the second cycle for first strand cDNA synthesis reaction. Affymetrix GeneChip Human Gene 1.0 ST arrays were hybridized with 5.5 µg of labeled sense DNA, washed and stained according to the Affymetrix GeneChip Expression Analysis Manual. Arrays were subsequently scanned on a GCS3000 7G Scanner. Data capture and initial array quality assessment were performed with the Affymetrix GeneChip Command Console v2.0 software.

Affymetrix Human Genome 1.0ST Arrays (Affymetrix, Santa Clara, CA, USA) covering 28,869 transcripts in the human genome were employed for transcriptional profiling and performed by Affymetrix. Three to four pre-ameloblast and secretory ameloblast samples collected by LCM were pooled for each array. RNA samples were extracted and purified by RNeasy Mini kits (Qiagen and Rnase-Free DNase Set) as described previously [63 (link)]. A Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used to determine total RNA concentrations, and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evaluate RNA quality [64 (link)] Both RNA samples were of acceptable quality (Average RIN of 9.0 and 8.0 for PABs and SABs, respectively), and there was no significant difference between their qualities. For each replicate, 100 ng of total RNA was amplified and labeled using the Affymetrix Whole-Transcript (WT) Sense Target Labeling Protocol without rRNA reduction. Array hybridization, washing, and scanning were performed according to the protocol described in WT Sense Target Labeling Assay Manual.

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.