title: GSM6514659: KGW2_TH_EPCAM_45; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue Digestion: Human fetal thymus and lung samples (pcw 16 -18) were mechanically dissected into <2 mm3 pieces using surgical scissors. Human fetal bronchus and esophagus 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 per tube using a tissue dissociator (gentleMACS, C tube, program m_spleen_02 (Miltenyi), 1-3 runs depending on tissue). Tissue fragments were transferred to a 50ml tube and incubate at 37°C for 20 min. Cells were pelleted at 110g, RT for 2 min and resuspended in 10 ml PBS. For persisting fragments, the procedure was repeated 1-2 times to yield single cells.  Cells were resuspended in MACS buffer (PBS, 1 %BSA, 2mM EDTA), filtered through a 100um cell strainer and counted. Epithelial Cell Enrichment: For fetal thymic tissue (16-18 pcw) only, cell suspension was depleted of thymocytes and other immune cells using Human CD45 MicroBeads (Miltenyi) and LD depletion columns (Miltenyi),10e8 cells/column, according to the manufacturer's instructions. Single cell suspensions of all tissues, (CD45-depleted thymus, bronchus, lung, and esophagus) 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 flowcytomery on Beckman FACSAria II with CD45-APC, CD3-FITC, CD205-PE, EpCAM-APC, and L/D-NearIR-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.   Gel-Bead in Emulsions (GEMs) were generated from single cell suspensions using the 3′ V3 chemistry and Chromium microfluidics system (10X Genomics). After reverse transcription, barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was subjected to 0.6x SPRI beads cleanup (Beckman, B23318). 25% of the amplified cDNA was subjected to enzymatic fragmentation, end-repair, A tailing, adaptor ligation and 10X specific sample indexing as per manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) analysis. Libraries were pooled and sequenced on an NovaSeq 6000 instrument (Illumina) using the recommended sequencing read lengths of 28 bp (Read 1), 8 bp (i7 Index Read), and 91 bp (Read 2).