title: GSM7847376: iSureHadCre_Postnatal HTO; Mus musculus; RNA-Seq
library_construction_protocol: To isolate ECs for scRNAseq experiments, livers and hearts were dissected, minced, and digested for 30 min with pre-548 warmed (37C) dissociation buffer (2.5mg/ml collagenase I (Thermo Fisher 17100017 ), 2.5mg/ml dispase II (Thermo Fisher 17105041), 1ul/ml DNAse in PBS containing Ca2+ and Mg2+ (Gibco)). The digestion tube was agitated every 3-5 minutes in a water bath. At the end of the 30 minutes incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca2+ or Mg2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450g, 5 min, 4°C). The supernatant was aspirated, and cell pellets were resuspended in 2ml 1x RBC lysis buffer (BioLegend, 420301) and incubated for 5 min on ice. To each sample were added 6 ml of sorting buffer, and samples were then passed through a 70um filter. Live cells were counted in a Neubauer Chamber using trypan blue exclusion. Cells from each condition were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 200ul antibody incubation buffer (PBS + 1% BSA) containing 1 μl CD31-APC and 1μl of hash tag oligo (HTO) conjugated antibodies (Biolegend). HTOs were used to label and distinguish the different samples when loaded on the same 10x genomics port, and in this way also guarantee the absence of batch effects. After the antibodies incubation, samples were transferred to 15 ml Falcon tubes, 1.4 ml sorting buffer was added, and samples were centrifuged (400g, 5min, 4°C). The supernatant was aspirated, pellets were resuspended in 1.5 ml sorting buffer and transferred to Eppendorf tubes, and the resulting suspensions were centrifuged (400g, 5min, 4°C). The resulting pellets were resuspended in 200 μl sorting buffer containing DAPI. Cells were sorted by FACS with an Aria Cell Sorter (BD Biosciences) using a 100um Nozzle 20 PSI and 16 32 0 or PURITY, Flow rate 1 - to preserve cell viability; ECs were identified as CD31+, MbTomato+ cells. Single cell suspensions of freshly isolated ECs were resuspended in Cell Capture buffer to a final concentration of 800 to 1600 cells/μl. scRNA-seq libraries were prepared using the Chromium Single Cell 3´ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions.