GSM7729685: Fetal Thymocytes 1; Homo sapiens; RNA-Seq
Human fetal thymus and lung samples (pcw 16 -18) were mechanically dissected into <2 mm^3 pieces using surgical scissors. Human fetal bronchus samples (pcw 16 -18) were opened longitudinally and mechanically scraped with scalpel to harvest the inner epithelial layer of bronchus and esophagus, respectively. All tissue samples were enzymatically digested with DNAse (10 mg/ml) and Liberase (2.5 mg/ml, Sigma Aldrich) per 10 g tissue using a tissue dissociator (gentleMACS, C tube, program m_spleen_02 (Miltenyi), 1-3 runs depending on tissue). For fetal thymic tissue (16-18 pcw) only, thymocytes were isolated from cell suspension using Human CD45 MicroBeads (Miltenyi) while other immune cells were depleted using LD depletion columns (Miltenyi),108 cells/column, according to the manufacturer's instructions. Single cell suspensions of all tissues, (CD45-depleted thymus, bronchus and lung) were enriched for epithelial cells using Human CD326 (EPCAM) Microbeads and LS positive selection columns (Miltenyi) according to the manufacturer's instructions. Quality control for thymocyte depletion and epithelial cell enrichment for thymic single cell suspensions was performed by flow cytomery on Beckman FACSAria II with CD45-APC, CD3-FITC, CD205-PE, EPCAM-APC, and Live Dead-Near InfraRed-APC-Cy7 prior to MACS purification. Due to epitope blockage from magnetic bead antibodies, additional markers, i.e. CD7-AF647 (thymocytes) and CD105-PerCP/Cy5.5 (thymic mesenchymal cells) were used to assess post enrichment purity. Total RNA was extracted from thymocytes and EPCAM positive selected epithelial cells using the Qiagen RNeasy kit. Takara SMART-Seq v4 Ultra low Input RNA kit 500pg-5ng of total RNA input were used followed by pooling of 2-12 samples into Hiseq4000 and NovaSeq6000 lanes at a 2 x 100 bp read length.