title: GSM8630762: 5 Days Postnatal Thymus Thymocytes; Homo sapiens; RNA-Seq
library_construction_protocol: Human fetal thymus samples, collected at postnatal day 5, 3 months, and 4 months, were mechanically dissected into fragments smaller than 2 mm³ using surgical scissors. Each sample was then enzymatically digested in a digestion medium (PBS) containing DNase (0.095 mg/mL) and Liberase TM (0.095 mg/mL, Sigma-Aldrich) on a rotating device to ensure continuous mixing. After 2–3 rounds of digestion (25 minutes per round), the cell suspension was centrifuged at 110 G for 2 minutes. The supernatant was collected for staining of thymic stromal immune cells prior to sorting.  The remaining cell pellet, containing epithelial cells, underwent additional rounds of digestion if necessary until there were no visible fragments. This cell suspension was then subjected to density centrifugation using Histopaque-1077 (Sigma-Aldrich) to enrich for epithelial cells in the low-density fraction (LDF). Cells from the LDF were then processed to deplete hCD45+ cells using Human CD45 MicroBeads/LD depletion columns (Miltenyi Biotec). Immune cells were stained using the following antibodies: CD19/20-BV650, CD5/CD7-FITC, CD123-PE, CD1c/CD11c-PE-Cy7, CD45-APC, and HLA-DR-APC-Cy7. Immune cells were sorted on a BD FACS Aria II SORP as follows: Thymocytes (CD45+, CD5+, CD7+), B Cells (CD45+, CD19+, CD20+), mDC (CD45+, CD1c+, CD11c+, HLA-DR+), and pDC (CD45+, CD123+, HLA-DR+). Epithelial cells were stained using the following antibodies: CD205-PE, HLA-DR-PE-Cy7, EPCAM-APC, and CD45-APC-Cy7. Epithelial cells were sorted as follows: cTEC (EPCAM+, CD205+, HLA-DR-, CD45-), mTECI (EPCAM+, CD205-, HLA-DR-, CD45-), and mTECII (EPCAM+, CD205-, HLA-DR+, CD45-). Cell viability was assessed using 7-AAD staining. Total RNA was extracted from sorted cells using the Qiagen RNeasy Kit. Illumina Stranded mRNA Prep 50-200ng Total RNA input were used to construct libraries followed by pooling libraries into NovaSeq6000 lanes at 2 x 100 bp read length.