GSM7295043: HOM_B3: FACS sorted endothelial cells from SOX7ECKO at 10.5dpc; Mus musculus; RNA-Seq
To validate the Sox7iECKO E10.5 RNA-Seq, staged matched (31-33ss) sibling controls and Sox7iECKO embryos were harvested from pregnant dams treated as specified with tamoxifen at E9.5. Whole embryos were subjected to single cell isolation using 0.071 mg/ml liberase in PBS buffer. CD31+ endothelial cells were isolated using Alexa 488 conjugated rat anti-CD31 (1:800, 563607, BD Pharmigen), live/dead cells were detected by 7AAD (1:1000, Invitrogen). Cell sorting was performed on a BD FACSAria Cell Sorter. RNA was harvested using Trizol (Thermo-Fisher) and purified with DirectZol RNA microprep kit (R2060, Zymo research). RNA-Seq libraries were prepared from polyA selected RNA and cDNA generated by the Smart-Seq2 protocol using the Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096). For each sample, 0.5 ng of cDNA was used as input for the Nextera XT library prep, with 12 cycles of PCR used to amplify the final library. The final Nextera XT libraries were quantified on the Agilent Bioanalyzer with the High Sensitivity DNA kit (Agilent Technologies, 5067-4626). Libraries were pooled in equimolar ratios.