title: GSM4063668: siRNA CYP2J2 rep_1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell culture materials including adult derived primary human cardiomyocytes (cat #36044-15), media (complete growth media, cat #M36044-15S), and extracellular matrix pre-coated flasks and plates (cat #E36044-15) were obtained from Celprogen Inc. (San Pedro, CA, USA). Media were further sterile filtered using a vacuum filter through a 0.22 μm polyethersulfone (PES) filter. Celprogen's protocols were followed for culture and passage of adult human ventricular cardiomyoctes. Experiments were performed between passages 6 and 8, where passage numbers were determined by number of times cells were treated with trypsin since receipt from Celprogen which was designated passage 1 for the purposes of these experiments. Briefly, cells were cultured and expanded using Celprogen pre-coated flasks and complete growth media with serum. Cells used for RNA isolation and subsequent experiments were harvested by washing the cells still attached to the plate with 1X PBS. After washing, all liquid was aspirated from the wells and the entire plate was stored in -80 °C until RNA extraction and purification. CYP2J2 gene expression was silenced using the RNAiMAX lipofectamine (Thermo Fisher Scientific, Waltham MA) and the CYP2J2 Trilencer siRNA or scrambled siRNA (Origene, Rockville, MD), using the manufacturer's suggested protocols. Silencing of the HPRT1 gene using the Origene siRNA served as positive control. Lipofectamine was delivered using a reverse transfection protocol.  siRNA was reconstituted to a stock concentration of 20 μM per the manufacturer protocols and prepared with the lipofectamine using OptiMEM reduced serum media (Thermo Fisher Scientific, Waltham, MA) by diluting to a concentration of 50 nM. Cells were washed with warm (37 °C) PBS and harvested using trypsin. Following trypsinization, cells were pelleted and resuspended and diluted to a concentration of 200,000 cells/mL in complete media. The lipofectamine/siRNA stocks were added to each well to a final concentration of 10 nM siRNA (250 μL volume of 50 nM siRNA/lipofectamine in OptiMEM), followed by the cells (1 mL of cell suspension) for a final volume of 1250 μL in each well. The cells were incubated with the lipofectamine/siRNA for 72 hours. Total RNA was extracted using the MagMax 96 Total RNA Isolation kit (Thermo Fisher Scientific, Waltham MA). Initial RNA quality (A260/A280) and quantity was determined using a Synergy HTX Multi-Mode Reader (BioTek, Winooski VT). RNA libraries were prepared for sequencing using standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM4063668