GSM9104349: HS2; Homo sapiens; RNA-Seq
Fresh skin samples were immediately washed, minced, and digested at 37°C for one hour to prepare single-cell suspensions using a composite enzyme solution with the following components:3.5 mg/ml Collagenase II, 1 mg/ml Collagenase IV, 0.02 mg/ml DNase I, 30 U/ml Hyaluronidase, 30 U/ml Keratinase, 400 µg/ml Liberase, 1.2 units/ml Dispase II. Following red blood cell lysis, AO/PI (Acridine Orange/Propidium Iodide) staining and trypan blue microscopy were performed for cell counting. Flow cytometry was performed to assess the proportion of CD45⁺ cells. 10 µL of CD45 antibody (555482; 25ug/ml; BD Biosciences) was added to the suspension for incubation, followed by cell sorting using a flow cytometer (BD FACS Aria III). Data were analyzed using BD FACSDiva 9.0.1 software. ScRNA-seq was submitted for Shanghai Genechem Co., Ltd. Single-cell library sequencing was conducted on the Illumina HiSeq X Ten, with 150 nt paired-end sequencing. The Chromium instrument and the Single Cell 3' Reagent kit V3.1 (Dual Index) were used to prepare individually barcoded single-cell RNA-Seq libraries following the manufacturer's protocol (10X Genomics). Briefly, sample partitioning and molecular barcoding were done on the Chromium Controller (10x Genomics), where we loaded cellular suspensions together with the Single-cell 3' Gel Beads on a Single-cell 3' chip, in which gel beads in emulsion (GEM) generation took place. RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell reagent kit V3.1 (10X Genomics). Sequencing was performed with Illumina NovaSeq.