GSM8305764: SH045, replicate 2, scRNAseq; Mus musculus; RNA-Seq
Tissue was washed with 1x PBS (phosphate-buffered saline, Gibco, cat. nr. 10010-23) and dissociated with ophthalmic scissors to pieces of 1-2 mm. The pieces were digested in 2 ml sCellLive™ Tissue Dissociation Solution (Singleron Biotechnologies, cat. nr. 1190062) at 37°C for 15 minutes in a 15-ml centrifuge tube (Sarstedt, cat. nr. 62.5544.003) with continuous agitation on a thermal shaker. The state of dissociation was checked at regular intervals under a light microscope. Following digestion, the suspension was filtered using a 40-µm sterile strainer (Greiner, cat nr. 542040). The cells were centrifuged at 350xg for 5 minutes at 4°C and the cell pellets were resuspended in 1 ml PBS. Cells were stained with a 0.4% w/v solution of Trypan Blue (Gibco, cat nr. 15250-061) and the cell number and viability were calculated in a hemacytometer under a light microscope. The single cell RNA-seq libraries were constructed using (GEXSCOPE™ Single Cell RNAseq Library Kit, Singleron Biotechnologies, cat nr. 4161031) according to manufacturer´s instructions. Briefly, for each library, the concentration of the single-cell suspension was adjusted to 3x105 cells/ml with PBS and the suspension was loaded onto an SD microfluidic chip to capture 6000 cells. Paramagnetic beads conjugated to oligodT probes that carry a unique molecular identifier (UMI) and a barcode unique to each bead (from the same kit) were loaded, after which the cells were lysed. The beads bound to polyadenylated mRNA were extracted from the chip and reverse transcribed into cDNA at 42°C for 1.5 hours, and the cDNA amplified by PCR. The cDNA was then fragmented and ligated to indexed Illumina adapters. The fragment size distribution of the final amplified library was obtained on an Agilent Fragment Analyzer.