GSM8448921: Epas1 EC-KO mouse 2; Mus musculus; RNA-Seq
Mice were given intraperitoneal injections of 100 μl pentobarbital. The mouse chest was dissected, revealing the heart. The vasculature was perfused with 10 ml phosphate buffered saline (PBS) containing 2% heparin. The aorta was dissected and cleaned and the adipose and connective tissue surrounding the aorta were removed. To digest the aorta, (which included the aortic arch, ascending, descending, thoracic, and abdominal portions) the sample was placed in a 35mm dish containing 125 U/mL collagenase type XI, 60 U/mL hyaluronidase type 1-s, 60 U/mL DNase I, 450 U/mL collagenase type I and 0.5 mg/ml elastase in 2.5 ml of PBS, at 37ºC for 1h. After the first 10 mins of incubation, the adventitia was removed using forceps. Following the incubation, single cell suspensions from the digested aorta were prepared by pipetting the solution through a 40 μm cell strainer. Cells were washed and resuspended in FACS buffer (PBS with 1% BSA) and stained with CD31-AF488 and CD45-APC antibodies and TOPRO3. Alive, CD45-, CD31+ cells were sorted using a BDFACSMelody cell sorter (BD Biosciences) into single wells of 384-well plates containing reverse transcription primers, deoxyribonucleotide triphosphates, External RNA Controls Consortium (ERCC) RNA Spike-Ins, and mineral oil. scRNA-seq libraries were generated using the SORT-seq protocol (Muraro et al., 2016) a partially robotized method based on the CEL-Seq2 protocol (Hashimshony et al., 2016). Single cells were lysed at 65°C for 5 min, and second-strand mixers and reverse transcriptase were added to the wells using the Nanodrop II liquid handling platform (GC Biotech). The mRNA of each cell was reserve-transcribed, double-stranded complementary DNAs (cDNAs) from single cells were pooled, and in vitro transcription was performed for linear amplification, resulting in amplified RNA. The Illumina sequencing libraries were prepared using TruSeq small RNA primers (Illumina), and the DNA libraries were then sequenced paired-end at 75–base pair read length using the Illumina NextSeq 500 by Single Cell Discoveries (Utrecht, The Netherlands). The RNA yield of the amplified RNA and the quality and concentration of the final cDNA libraries were assessed by Bioanalyzer (Agilent).