GSM7696245: EC, tie1 Homo, replicate 1, RNAseq; Danio rerio; RNA-Seq
Trunk and tail region from 47 hpf Tg(kdrl:EGFP);Tg(lyve1:DsRed) tie1-/- zebrafish embryos with blood flow and their tie1+/? siblings were manually dissected and collected into a low binding 12 well plate. After washed in E3 media, samples were incubated with 1 ml of lysis solution containing collagenase P (Roche) and TrypLE Express Enzyme (Gibco) for 20 minutes under occasional pipetting. The dissociated cells in suspension medium (phenol red free DMEM with 1% FBS, 0.8 mM calcium chloride, 50 U/ml penicillin, and 0.05 mg/ml streptomycin) were subjected to cell sorting using a FACS Aria III cell sorter (BD Bioscience). EGFP and DsRed double positive cells were collected as venous and lymphatic endothelial cells for further RNA preparation. Total RNAs were prepared from kdrl:EGFP+/lyve1:DsRed+ ECs of tie1-/- embryos and their tie1+/? siblings using the NucleoSpin XS kit (Macherey-Nagel, 740902.50) according to the manufacturer's instructions. Reverse transcription and cDNA library preparation were performed with a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech, 634888). Libraries were prepared using an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7760)