GSM8594753: HSK, scRNAseq; Mus musculus; RNA-Seq
The fresh lung tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron) on ice after the surgery within 30 mins. The specimens were washed with Hanks Balanced Salt Solution (HBSS) 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 minutes. 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 minutes to remove red blood cells. The mixture was then centrifuged at 300 × g 4°C for 5 minutes to remove supernatant and suspended softly with PBS. For each experimental group, cells from 5 mice were pooled into one sample for single cell capture and cDNA library construction. Single-cell suspensions (2×10^5 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)13. Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.