GSM8635335: PC9,Gefitinib,rep 2; Homo sapiens; RNA-Seq
Cells were detached from culture dish with treatment of 2mL trypsin for 10cm2 dish. After counting and checking viability, single cell suspension were prepared at concentration of 300,000 cells/mL in PBS containing 10% Optiprep. The total RNA library was prepared in two steps. First, total RNA was captured in nanodroplets, and single-cell barcodes were assigned to reversely transcribed cDNA. Subsequently, the cDNA was pooled for in vitro amplification and integration of Illumina adapters. Briefly, using a microfluidics device, single cells were encapsulated in water-in-oil droplets containing a lysis buffer with reverse transcription (RT) enzymes and hydrogel beads carrying primers. These primers included single-cell barcodes and rDS/polyT sequences, as well as the Illumina adapter PE1 and a T7 promoter for in vitro transcription (IVT). Following reverse transcription, the cDNA from all droplets was pooled, unused primers were removed, and the cDNA was cleaned. The second strand was then synthesized prior to IVT amplification. The amplified RNA was slightly fragmented, and the Illumina adapter PE2 was integrated via a random RT step. Finally, the library was amplified by PCR, during which Illumina indexes were incorporated.