GSM7996300: D_06, scRNA-seq; Homo sapiens; RNA-Seq
Fresh human decidual tissues were washed, cut into small pieces, and then enzymatically dissociated in RPMI 1640 medium (HyClone, Thermo Fisher Scientific, Waltham, MA, USA) containing 1 mg/mL type IV collagenase (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 0.01 mg/mL DNase I (Sigma-Aldrich, Louis, MO, USA) at 37 °C with constant agitation for 40 min. The suspensions were filtered through a 70-µm sterile strainer (BD Biosciences, San Jose, CA, USA) using a syringe plunger, washed, and centrifuged on a discontinuous gradient of 20%, 40%, and 60% Percoll bulk for 30 min at 800 g. The DICs were then immediately used for the subsequent cell sorting. Fresh human peripheral blood was collected in K2 EDTA tubes and processed within a few hours. An equal volume of room-temperature phosphate buffered saline (PBS) was mixed with blood and carefully layered over Ficoll Hypaque solution (MP Biomedicals, Santa Ana CA, USA). After centrifugation for 30 min at 2500 rpm (with no break), at room temperature, the mononuclear cell layer was washed three times with excess PBS for 10 min at 1500 rpm and resuspended in 2 ml of PBS. After cell counting, the PBMC samples were immediately allocated for subsequent cell sorting. Human Tregs (CD45+CD4+CD25hiCD127lo), Tconvs (CD45+CD4+CD25-) and monocytes/macrophages (CD45+CD14+) from decidua and matched peripheral blood were sorted on an Aria Cell Sorter (BD Biosciences). Three types of cells were pooled, counted, and loaded onto a 10x Genomics Chromium Controller instrument for single-cell encapsulation with a 5'VDJ kit (10x Genomics) to recover an estimated 5,000 cells per library. Reverse transcription was performed using a T100TM Thermal Cycler (Bio-Rad) according to the manufacturer's protocol, followed by the purification of cDNA with silane magnetic beads. Full-length V(D)J segments were enriched via PCR amplification with primers specific to the TCR constant regions. The enriched products were then subjected to Enriched Library Construction. Library quality and concentration were assessed using an Agilent Bioanalyzer 2100. Libraries were run on a Hiseq X or Novaseq for Illumina PE150 sequencing. Due to the unqualified cell viability after FACS-sorting, the decidual Tconvs of P01 and macrophages of P02 were not pooled and examined in the subsequent single-cell sequencing analysis. single-cell RNA-seq and TCR-seq