GSM5737376: tdTomato 1; Mus musculus; RNA-Seq
Evan's blue dye was administered intradermal into the mouse hindpaw to identify the two popliteal lymphatic vessels that run on either side of the superficial saphenous vein in the back of the calf draining into the popliteal lymph node behind the knee. All three vessels were dissected, manually and enzymatically digested (Accumax; Innovative Cell Technologies Cat# AM-105) for 1h at room temperature, filtered with a 75um strainer, and single cell suspensions were sorted by fluorescence activated cell sorting based on tdTomato fluoresence (tdT1 & tdT2) or scatter profile (WT and TNF). Sorted cells were then submitted to the Genomics Research Center at the University of Rochester for library preparation and sequencing. All cells in each sample were loaded on a Chromium Single-Cell Insrument (10x Genomics) and single-cell Gel Bead-in-Emulsions were generated. The Chromium Single-Cell 3' Library & Gel Bead Kit (10x Genomics) was used to generate the library based on the manufacturer's instructions. Barcoded cDNA from the Gel Bead-in-Emulsions was purified with DynaBeads MyOne Silane Beads (ThermoFisher Scientific Cat# PN37002D) with PCR amplification for 3' cDNA library construction. NextSeq 550 (tdT1) and NovaSeq 6000 (tdT2, WT, TNF) Illumina sequencers generated the sequenced data. RNA libraries were prepared for sequencing using standard Illumina protocols