GSM7050222: COV1_SM_CTL; Homo sapiens; RNA-Seq
RNA was extracted using the RNeasy plus mini kit (Qiagen). cDNA libraries for RNA-seq were prepared  by the Genomics Core at the Van Andel Institute. cDNA was synthesized from the isolated RNA samples using the SuperScript™ IV VILO™ Master Mix (11756050, Invitrogen) with ezDNase enzyme treatment according to the manufacturer's protocol. cDNA concentration was quantified and diluted to 1 µg/µL. Individual master mixes for each gene of interest were prepared using the TB Green® Premix Ex Taq II (TLi RNaseH Plus) reagent kit (RR820A, Takara Bio) and primer sets ordered from Sigma-Aldrich. Each master mix contained a 1X concentration of TB Green® Premix Ex Taq II (TLi RNaseH Plus), 0.4µM of forward and reverse primers, and nuclease-free water. To each well of a 96-well PCR plate (AB-0600-L, Thermo Scientific), 18 µL of the prepared master mix was added, followed by 2 µL of template cDNA. The reaction was carried out using the CFX96™ Real-Time System (Bio-Rad) using the following settings: a holding stage of 30 seconds at 95°C, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, and ending with the melt curve stage. The instrument software from Bio-Rad was used to quantify the results. Sequencing was performed by 2 × 150 bp paired-end read format.