GSM7844851: Htag2; Homo sapiens; RNA-Seq
Initially, the LLCs were detached by adding 0.5X TrypLE Select to the culture dishes and incubating at 37°C for 2 minutes. In accordance with the protocol, the cells were resuspended in 1X PBS and transferred into cartridges, along with beads. Subsequent to this, reverse transcription reaction and PCR were performed. Using the Rhapsody system, cells and beads were loaded. The cells were lysed to dissolve them, allowing the mRNA derived from each cell to be captured by barcoded beads. Following this, beads were collected, and cDNA was synthesized through reverse transcription. The mRNA that served as a template for cDNA was then degraded using RNase. Terminal transferase was employed to add a homopolynucleotide to the cDNA end using a single type of dNTP and terminated with the same type of ddNTP. A second strand was synthesized on the cDNA using a primer containing a single type of dNTP. This double-stranded DNA was then amplified via PCR, referred to as the first WTA. After PCR amplification, the sample underwent size selection, or purification, using beads. The size-selected sample was then amplified again using PCR, marking the second WTA. Following this, the sample was subjected to another round of size selection using beads. The size distribution of the sample was then measured using the MultiNA microchip electrophoresis device from Shimadzu Corporation.