GSM9335675: HD-wt2; Mus musculus; RNA-Seq
To isolate total cardiac ECs from Hand2f/f;VE-cad-CreERT2; iSure-Cre mice and iSure-Cre mice, we induced Hand2 deletion at E11.5-12.5, treated the mice with tamoxifen, and isolated the hearts at E14.5. HD-wt (iSure-Cre mice) and HD-ko (Hand2f/f;VE-cad-CreERT2; iSure-Cre) hearts were dissected, minced, and digested in a cell-dissociated solution. The cell pellet was resuspended and incubated with CD31-antibody (BD Pharmingen, 551262). Twice the volume of blocking solution was added to stop the reaction, and centrifugation was then performed. The cells were resuspended in the appropriate blocking solution and stored on ice prior to sorting. DAPI (1:1000) was then added to label the dead cells. A FACSAria III flow cytometer (BD Biosciences) was used for sorting. Approximately 9000-12000 DAPI-, APC-CD31+/Tomato+ ECs were sorted per sample for the subsequent experiments. Total RNA from sorted ECs was carried out using the RNeasy Micro Kit (Qiagen, 74004). Total RNA was extracted followed by library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina®). Agilent 4200 bioanalyzer was employed to evaluate the concentration and size distribution of cDNA library before sequencing with an Illumina novaseq6000. The protocol of high-throughput sequencing was fully according to the manufacturer's instructions (Illumina).