title: GSM8027886: Pre-treatment replicate3, scRNA; Mus musculus; RNA-Seq
library_construction_protocol: The fresh tissues were stored in the Primary Tissue Storage Solution (bioGenous) on ice after the surgery within 30 mins. The specimens were washed with PBS (HyClone) for three times, minced into small pieces, and then digested with 3 mL sCelLiveTM Tissue Dissociation Solution (Singleron) by Singleron PythoN™ Tissue Dissociation System at 37 °C for 15 min. The cell suspension was collected and filtered through a 40-micron sterile strainer. Afterwards, the GEXSCOPE® red blood cell lysis buﬀer (RCLB, Singleron) was added, and the mixture [Cell: RCLB=1:2 (volume ratio)] was incubated at room temperature for 5-8 min to remove red blood cells. The mixture was then centrifuged at 300×g, 4 ℃ for 5 mins to remove supernatant and suspended softly with PBS. GFP+ RENCA cells were isolated using FACS. Finally, the samples were stained with Trypan Blue and the cell viability was evaluated microscopically. Single-cell suspensions (2×105 cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.