HAECs were transfected by control siRNA or TERF2IP siRNA, and after 48 hrs of transfection HAECs were exposed to disturbed flow (d-flow). After 24 hrs of disturbed flow RNA was extracted for futher microarray processing.
Growth protocol
HAECs were cultured in Petri dishes or flasks coated with 0.2% gelatin type A (#901771; MP Biomedicals, Santa Ana, CA, USA), in Endothelial Cell Medium (ECM, #1001, ScienCell, Carlsbard, CA. USA) containing 465 mL of basal medium, 25 mL of fetal bovine serum (FBS, #0025, ScienCell, Carlsbard, CA, USA), 5 mL of Endothelial Cell Growth Supplement (ECGS, #1052, ScienCell, Carlsbard, CA, USA) and 5 mL of penicillin/streptomycin solution (P/S, #0503, ScienCell, Carlsbard, CA, USA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by using an RNeasy Plus Mini Kit (catalog #74134; QIAGEN) according to the manufacturer's instructions.
Label
biotin
Label protocol
Fragmented and biotinylated labeled ss-cDNA were prepared according to standard Affymetrix WT Plus protocol (P/N 703174) from 100 ng total RNA
Hybridization protocol
Following fragmentation and labeling, 5.2 ug of labeled ss-cDNA were hybridized for 16 hrs at 45 degree on GeneChip Mouse Transcriptome arrays. Arrays were washed and stained in Affymetrix Fluidics station 450.
Scan protocol
Arrays were scanned using Affymetrix GCS3000 scanner
Data processing
The array data were incorporated into the Transcriptome Analysis Console 3.0 software program (Affymetrix), and GeneChip data were normalized using Tukey’s biweight average algorithm and represented as a biweight average on a log2 scale. A principal component analysis and differential profile (heatmap) of the d-flow responses in TERF2IP-depleted cells were analyzed using Qlucore Omics Explorer version 3.2. The principal component analysis was performed at a stringent p = 3.3e-5 using ANOVA on the basis of TERF2IP siRNA and d-flow treatment.
