GSM4804695: GC3-LN10; Homo sapiens; OTHER
Tissues were digested for 60 minutes at 37°C and then pipetted up and down every ten times every 10 minutes. The tissue suspensions were then filtered with a 70um nylon mesh (ThermoFisher Scientific, USA) and were centrifuged at 450g for 5 minutes. Pellets were resuspended for live cell staining using CFSE incubation for 5 minutes Single cells from each tissue were manually picked under fluorescent microscopy (X71, Olympus, Japan) using mouth pipette. Each of the harvested single cells was transferred into 2uL of CLB (cell lysis buffer) in 0.2mL PCR tubes. Libraries of isolated single cells were then prepared as per the Smart-seq2 protocol15 with modifications on reverse transcription and amplification cycles.  Oligo-dT primed RT (reverse transcription) was performed by Smartscribe (Takara, Japan) reverse transcriptase and locked TSO oligonucleotide (Exiqon, Danmark) upon single cells. Full-length cDNA amplification was conducted by PCR amplification for 22 cycles with Hifi HotStart ReadyMix reagent (KAPA Biosystems, USA) and purified by 0.6x AMPure beads (BD, USA). Barcoded libraries were fragmented and segmented with Library Prep kit (Nextera XT, Illumina, USA). Pooled libraries with unique N5-N7 barcodes were sequenced with a Hiseq 2500 sequencer (Illumina, USA) and 50SE reads flow cell.