GSM6910166: ESG1, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
Healthy human skins were dissected and placed on a sterile 30-mm culture dish with ice-cold PBS. Tissues were then transferred into RPIM medium, minced with scissors, and placed in a 30-mm culture dish containing neutral red working buffer (10 uM neutral red). We isolated whole ESGs under a stereomicroscope using forceps and collected ESGs into a 12-well cell culture dish containing 2.5 mg/ml librease TL in RPIM medium and incubated at 37 ̊C for one night. The single-cell suspension was obtained the next day by repeatedly aspirating and dispensing the solution with a 10-ml pipette for 3 min with 3 ml warm 0.05% trypsin / 0.5M EDTA. Following tissue digestion, 40 ml of resuspension media (RIPM supplemented with 5% normal human serum and 1% pen/strep) was added to neutralize enzyme activity. The digestion mixture was passed through a 70 mm cell strainer and centrifuged at 150 x g for 8 min at 4 degrees. The pellet was resuspended and incubated in dead cell remove buffer to remove dead cells using the MS columns of the Dead Cell Removal Kit as per the manufacturer's directions. Finally, the cell pellet was resuspended in 1 ml of 0.04% BSA in DPBS. This final cell suspension solution was passed through a 40 mm cell filter to discard debris. Cell number was counted using the automated cell counter Countess. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, ~8000 ESG cells for sample-1, ~10000 ESG cells for sample-2 and sample-3 were mixed with the retrotranscription reagents and pipetted into a Chip A Single Cell, also containing the Single Cell 3' Gel Bead suspension and Partitioning Oil. The Chip was subsequently loaded into a Chromium Single Cell Controller. The cells were captured in nanoscale droplets containing the reagents needed for reverse transcription and a gel bead. Following the manufacturer's protocol, the resulting gel bead-in-emulsions (GEMs) were then transferred to a thermocycler to perform the retrotranscription. Each gel bead contained a specific 10X Genomics barcode, an Illumina R1 sequence, a Unique Molecular Identifier (UMI), and a poly-dT primer sequence. Following an amplification step, cDNA was further processed by fragmentation, end-repair, and A-tailing double-sided size selection. Finally, Illumina adaptors and a sample index were added through PCR using a total number of cycles adjusted to the cDNA concentration. After sample indexing, libraries were subjected to double-sided size selection, and quantification was carried out using the Qubit dsDNA HS Assay Kit, and cDNA integrity was assessed using D1000 ScreenTapes. Library quality control metrics were as follows: 550.19 pg/µl with an average size of ~454 bps. Paired-end sequencing (20,000 reads / cell) was finally performed with MGISEQ G400RS platform (MGI).