GSM5049686: Sample D705; Homo sapiens; RNA-Seq
Twenty-four hours post-transfection, cells were washed twice and harvested in 1 × PBS, and Total RNA was extracted from all surviving cells using RNeasy Plus Mini Kit (74136, QIAGEN). We treated the mRNA with RapidOut DNA Removal Kit (K2981, Thermo Scientific) to remove the trace amount of genomic DNA residue according to the product manual. The purified RNA was then subjected to reverse transcription with High-Capacity cDNA Reverse Transcription Kits (4374967, Applied Biosystems). Briefly, 1.5μg RNA was added into 10μl of 2× RT Master Mix and made up to the final 20μl with nuclease-free water. The reactions were incubated at 25°C for 10 min, followed by 120 min at 37°C, then were inactivated Reverse Transcriptase by heating to 85°C for 5 min. The cDNA products were stored at -20°C or -80°C and ready for qPCR analysis and preparing the NGS sequencing library. RNA libraries were prepared for sequencing using standard Illumina protocols