GSM8880122: Retinal EC, P6, Early G1, rep2; Mus musculus; ATAC-seq
Mice at age P6 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. Endothelial cells were sorted to isolate 3 samples of early G1 (blank fluorescence) cells and 4 samples of late G1 (red fluorescence) cells, then nuclei were isolated as described for single cell ATAC sequencing preparation. Isolated nuclei were incubated in Transposition Mix containing 25 µL TD Buffer, 2.5 µL TDE1 enzyme, 0.5% Digitonin, and 22.5 µL H2O (items from Nextera XT DNA Library Preparation Kit) at 37°C for 30 min. DNA was purified using a MinElute PCR Purification Kit. Tagmented DNA samples were PCR amplified with unique barcoded primers for demultiplexing after sequencing. PCR products were purified using MinElute PCR Purification Kit and cleaned with AMPure Beads. Samples were submitted to Novogene for next-generation sequencing.