GSM8880129: Retinal EC, P15; Mus musculus; ATAC-seq
Mice at ages P6 and P15 were euthanized according to approved protocols, and retinal tissue was immediately harvested from isolated eyes by microdissection removal of cornea, adventitial, and vitreous body tissue. Retinal tissue was then enzymatically digested in a digestion solution containing 1.0 mg/mL Collagenase Type II and 10% FBS in DMEM for 20 min at 37°C. Digested cells were washed and resuspended in staining buffer containing 10% FBS, 20 mM HEPES, and 1.0 mg/mL D-Glucose in HBSS with fluorescently-conjugated anti-CD31 and anti-CD45 antibodies. Immuno-stained cells were washed, resuspended in FACS buffer containing 1% FBS in PBS, stained with Propidium Iodide (PI), and sorted by FACS using a FACSMelody sorter (BD Biosciences) to isolate the PI-CD31+CD45- population as endothelial cells. Isolated endothelial cells from P6 and P15 murine retinal tissue were pelleted at 300 rcf for 5 min and resuspended in 50 µL Cell Buffer containing 0.04% BSA in PBS. Cells were re-pelleted and resuspended in 50 µL Lysis Buffer containing 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, and 1% BSA in Nuclease-free water, then mixed by pipetting and incubated on ice for 5 min. Then, 50 µL chilled Wash Buffer containing 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and 1% BSA in Nuclease-free water was added, and extracted nuclei were pelleted by centrifugation at 500 rcf for 5 min. Supernatant was removed and 45 µL chilled Diluted Nuclei Buffer containing a 1x dilution of Nuclei Buffer 20x (from 10x Chromium Next GEM Single Cell ATAC Library and Gel Bead Kit) in Nuclease-free Water was added. Nuclei were pelleted, supernatant was removed, and 7 µL chilled Diluted Nuclei Buffer was added. Nuclei were immediate used in the Chromium Next GEM Single Cell ATAC Kit v1.1 protocol with subsequent quality control and sequencing according to validated standard operating procedures established by the University of Virginia Genome Analysis and Technology Core, RRID:SCR_018883, using a 10x Chromium Controller and Illumina NextSeq 2000.