GSM6944094: E15, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Single-cell suspension of endothelial cells were prepared as described previously (Pinto et al., 2013, PMID 23578979). C57Bl6 8 weeks old adult animals, P2 postnatal, E15 and E12 were used for single-cell suspension preparation. Adult animals were sacrificed by cervical dislocation while embryonic and postnatal animals were decapitated. Adult and postnatal hearts were chopped with a scalpel until getting 1-2mm pieces of tissue. The enzymatic digestion was adapted to every stage analyzed: for E12, 20min of Collagenase/Dispase (Sigma-Aldrich) (1mg/ml), for E15, 30min of Collagenase/Dispase (1mg/ml), and for P2 and adult hearts, 20 min of Collagenase II (500units/ml) (Worthington) plus a second digestion step with Collagenase/Dispase for 30min. All steps were performed at 37ºC, under agitation and pippeting up and down the solution within the tubes every 5min. To stop the enzymatic reaction 10ml of cold FACs buffer (HBSS/2%FBS/10mM Hepes) was added to the samples and then spinned down (400G, 5 min). For adult samples, red blood cells lysis step (Invitrogen) was performed. After enzymatic digestion, cell debris removal kit (Miltenyi) was performed and finally, all suspension filtered through a 40um strainer.  Single-cell suspensions were incubated with primary antibodies in FACs buffer for 20 min on ice. The following antibodies were used: APC-conjugated anti-CD31 (Thermo Fisher, 17-0311-80) and PE-conjugated anti-CD45 (BD Biosciences, 553081). Samples were centrifuged (400G, 5 min) and resuspended on FACs buffer and stored on ice until applied to the flow cytometer. CD31+/CD45- cells were sorted on an Aria II into 1.5ml tubes with EBM2 media. Compensation controls were set up for each single channel. After completion of sorting, 10% DMSO was added and cell suspension frozen at -80ºC. Several rounds of single-cell suspension preparation, sorting and freezing was repeated for embryonic samples in order to get a significant number of cells: 8 weeks old adult animals (n=6), P2 postnatal (n=21), E15 (n=48) and E12 (n=37).  Frozen vials of sorted CD31+/CD45- cells from E12, E15, P2 and 8wo adult hearts were thawed in a water bath at 37ºC. For E12 and E15, a dead cell removal kit (Miltenyi) was applied. For P2 and adult, re-sorting by flow cytometry and selection of live cells was performed. 3′ gene expression libraries were prepared according to the manufacturer's instructions of the v2 Chromium Single Cell Reagent Kits (10X Genomics). Quality control of cDNA and final libraries was done using Bioanalyzer 2100 (Agilent).