title: GSM7676123: KBTRE, normal, replicate S4; Danio rerio; RNA-Seq
library_construction_protocol: For tissue disaggregation, whole embryos were incubated with a digestion cocktail (10mg/ml collagenase; 5mg/ml hyaluronidase; and 20mg/ml proteinase K and constant mechanical dissociation was exerted for 10 minutes. Cells suspension were filtered through a 40 µm nylon mesh and L-15 medium was added to stop the digestion process. Isolated cells were pelleted, resuspended in L-15 medium and processed by FACS. Red+ and green+ cells were loaded into the Chromium Controller instrument to generate Gel beads in EMulsion (GEMs). GEM-RT was performed in a C1000 Touch Thermal cycler. After reverse transcription, GEMs were broken, and the single-strand cDNA was cleaned up using DynaBeads MyOne Silane Beads and the SPRIselect Reagent Kit. cDNA was amplified using the C1000 Touch Thermal cycler and cleaned up with the SPRIselect Reagent Kit. Libraries were constructed using the GemCode Single-Cell 3' Library Kit following these steps: 1) end repair and A-tailing, 2) adapter ligation, 3) postligation cleanup with SPRIselect, 4) sample index PCR and cleanup. The barcode sequencing libraries were quantified by quantitative PCR.