Tn5 transposase and transposase reaction buffer

RNA was extracted from an aliquot of dissociated tissue prior to filtering and antibody staining and from GFP-positive and GFP-negative FACS sorted cells using the RNeasy Micro Plus kit (74034, QIAGEN, Venlo, Netherlands). DNA for MethylC-seq was prepared from GFP-positive FACS-sorted cells using the DNeasy Blood and Tissue kit (69504, QIAGEN). For ATAC-seq,~50,000 GFP-positive FACS-sorted cells were resuspended in ice-cold lysis buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Tricine-KOH, 0.1% Igepal CA-630) and immediately centrifuged at 500 x g for 10 min at 4°C to prepare nuclei. The resulting nuclear pellet was resuspended in a 50 ul reaction volume in Tn5 transposase and transposase reaction buffer (FC-121–1030, Illumina Inc., San Diego, CA) and the tagmentation reaction was incubated at 37°C for 30 min.

For acutely isolated ECs, RNA was extracted from GFP-positive and GFP-negative cells that were FACS sorted directly into QIAGEN Buffer RLT Plus and then processed using the RNeasy Micro Plus kit (74034, QIAGEN, Venlo, Netherlands). For cultured ECs, RNA was extracted by adding QIAGEN Buffer RLT Plus directly into the well followed by extraction using the RNeasy Micro Plus kit. For ATAC-seq, ~50,000 GFP-positive FACS-sorted cells or trypsinized cultured ECs were gently centrifuged and then resuspended in ice-cold lysis buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Tricine-KOH, 0.1% Igepal CA-630) and immediately centrifuged at 500 x g for 10 min at 4°C to prepare nuclei. The resulting nuclear pellet was resuspended in a 50 ul reaction volume in Tn5 transposase and transposase reaction buffer (FC-121–1030, Illumina Inc, San Diego, CA), and the tagmentation reaction was incubated at 37°C for 30 min.