GSM9335665: HD-wt2; Mus musculus; OTHER
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. Briefly, the ECs were incubated with ConA Beads Pro, resuspended with precooled primary HAND2 antibody (Abcam, ab200040, 1:20) or control IgG, and incubated overnight at 4 ℃. The next day, the samples were incubated with a secondary antibody (Vazyme, AB207-01) and Hyperactivep A/G-TransposonPro reagent. Cells were resuspended in Trueprep Tagment Buffer L and incubated at 37 °C for 1h. Low amounts of E. coli DNA carried through the protocol were used for optional spike-in normalization. DNA fragments were extracted using the DNA Extract Beads Pro kit. Library construction was performed according to the manufacturer instructions. After passing quality control, the samples were subjected to high-throughput sequencing using the Illumina platform.