GSM8013670: day15,treat12,scRNAseq; Mus musculus; RNA-Seq
The skin tissues without separating dermis and epidermis were dissociated from the diabetic mice wound and washed with pre-cold PBS buffer. And skin tissues were minced as smaller as possible in digestive enzyme mixture and then incubated at 37°C for 1.5 h. Digestive enzyme mixture contains collagenase IV ( 200 mg/ml), Deoxyribonuclease I (1 mg/ml) , HEPES buffer solution (25mM) and RPIM-1640 medium supplemented with 10% fetal bovine serum. Subsequently, digestion process was terminated by adding the medium, and the digested skin tissues were further milled and filtered through a 40 μm strainer. Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent after removal erythrocytes (Miltenyi 130-094-183). Afterward, fresh cells were washed with ice-cold RPMI 1640 containing 2% FBS for twice and then resuspended at 1×106 cells per ml in 1×PBS and 0.04% BSA. Single-cell RNA-seq libraries were prepared using SeekOne® Digital Droplet (SeekOne® DD) Single Cell 3' library preparation kit V2.1.  Briefly, appropriate number of cells were mixed with reverse transcription reagent and then loaded to the sample well in SeekOne® DD Chip S3 (ChipS3). Subsequently, Barcoded Hydrogel Beads (BHBs) and partitioning Oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 42°C for 90 minutes and inactivated at 85°C for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally the indexed PCR were performed to amplified the DNA representing 3' polyA part of expressing genes which also contained Cell Bar code and Unique Molecular Index. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length