title: GSM9150344: Carotid Artery, CDH5cre negative Cx43fl/fl, 18hr post-injury; Mus musculus; RNA-Seq
library_construction_protocol: Both EC-Cx43 KO (CDH5cre positive) and EC-WT (CDH5cre negative)  mice were euthanized by CO2 overdose and cervical dislocation 18 hr post carotid EC injury and the injured carotid was exposed without perfusion to minimize time and disruption to the endothelial layer. The carotid was then removed from the mouse and dipped in a solution of heparin saline followed by a saline-only solution. Before digestion, carotids were stored in ice-cold EC media (Vascular Cell Basal Medium; ATCC #PCS-100-030) supplemented with all components of an Endothelial Cell Growth Kit (ATCC #PCS-100-041) excluding fetal bovine serum (FBS) while subsequent carotids were removed. To enrich for EC while collecting enough total cells for processing and sequencing, carotids were cut open lengthwise prior to removal to expose the EC layer for digestion. Carotids were kept whole and a short 20 min digest (described below) was performed.  Up to two carotids were digested in a 1 mL digestion solution of 0.2 mg/ml liberase TM (Roche #5401119001) and 60 U/ml DNase1 in supplemented EC media. Carotids were incubated at 37°C and 400 rpm in a benchtop orbital shaking heat block for 20 min, pipetting up and down with a 1 mL pipette every 5 min. Digestion was stopped with the addition of 1 mL stopping solution (2% FBS, 1mM EDTA, in supplemented EC media), after which samples of the same condition were combined (up to 5 at a time to reduce time to digestion). A 100 μm cell strainer was primed by flushing through 2 mL of supplemented EC media with 2% FBS. Cell solutions were filtered through the strainer and rinsed with 3 mL of supplemented EC media with 2% FBS. The flow through was centrifuged at 1000 rpm, 4°C for 10 min, followed by removal of the supernatant and resuspension of the cell pellet in 2 mL RBC lysis buffer (Invitrogen 00-4333-57) at room temperature for 5 min. RBC lysis was stopped using 10 ml supplemented EC media with 2% FBS, samples were centrifuged at 1000 rpm, 4°C for 10 min and the cell pellet was resuspended in 10% DMSO, 10% FBS in cold supplemented EC media. Single cell suspensions were slowly frozen at -80°C overnight in a cell freezer container, then transferred to liquid nitrogen until sequencing. A total of 15 carotids for EC-WT and 20 carotids for EC-Cx43 KO were pooled for use for scRNAseq by Novogene. Novogene scRNAseq Methods. Cell viability analysis, RNA integrity analysis, library preparation, sequencing, raw read count generation, and reference genome alignment were performed commercially by Novogene (Sacremento, CA, USA) using their Plant and Animal 10X Single Cell Transcriptome Sequencing pipeline. Full parameters, protocols, and quality control reports are available on request, but are explained briefly below:  Sample and Library Preparation. Cryopreserved cell samples were thawed and processed by Novogene. Cell count and viability were assessed using a TC20 Automated Cell Counter (Bio-Rad, USA) with both samples achieving approximately 60,000 cells with an overall cell viability of >75%. Libraries were then constructed using the 10x Chromium Single Cell 3' Kit with a target of 10,000 cells.  Sequencing and Data Preprocessing. Sequencing was performed on the NovaSeq X Plus Series (Illumina, USA) platform with 150 bp paired end reads with a minimum of 150 G raw data per sample.