GSM3690766: CN15_18_USR17005233L; Homo sapiens; RNA-Seq
At the day of harvesting the culture media was removed by aspiration and the cells were washed twice using 1 ml PBS each with aspiration. Subsequently, 600 µl of RLT Plus reagent was directly added into each well to homogenise cells in situ. Qiagen RNEasy plus kit was used for the extraction of total RNA according to the manufacturer's protocol. Extracted RNAs were eluted into 50 µl of RNA-free water and the concentration was determined by nanodrop. Approximately 5 µg total RNA was recovered from each well. Total RNAs isolated by using MirVana kit (Thermofisher) were used in RNA-seq. After QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Subsequently, terminal repair and sequencing adaptor ligation were applied to the templates. The double-stranded cDNA library is completed through size selection and PCR enrichment. Reverse-transcription was performed using ThermoFisher High-capacity RNA-to-cDNA reverse transcription kit with random hexamers according to the manufacturer's protocol. Briefly, 1 µg of total RNA was used for each 20 µl RT reaction. The reaction products were then diluted with 180 µl of RNase-free water for qPCR analysis. RNA quality control and gene expression quantification were performed by the qPCR method using gene-specific FAM-labelled TaqMan probes (ThermoFisher) against VPS45 with GAPDH as internal control on a Roche 480 II machine.