title: GSM3928161: EPC_GEO; Homo sapiens; RNA-Seq
library_construction_protocol: Fatty tissue was excised and remaining cardiac tissue was suspended in Basic Buffer (15 mL) and minced into 1 mm3 pieces. After mincing, tissue and Basic Buffer were collected in 50 mL Falcon tube. Digestive solution containing collagenase, type II 225 U/mg dry weight (Worthington, catalog #LS004174, Bio Corp, Lakewood, NJ) was dissolved in Basic Buffer (2-2.5 mg/mL) and incubated with tissue pieces for 1.5-2 hours at 37°C with continuous shaking. Digestion solution was refreshed at the one-hour time point and resulting suspensions were centrifuged at 350 g and resuspended in CPC media. Final suspension was filtered through a 100-µm filter (Corning, Inc., catalog #352360) followed by a 40-µm filter (Corning, Inc., catalog #352340) and centrifuged at 150 g for 2 minutes to collect CMs. The supernatant was collected and centrifuged at 350 g and resuspended in CPC media and incubated overnight at 37°C in 5% CO2 incubator. The following day, cells in suspension were collected in 50 mL Falcon tube. Any cells attached were dissociated using a 1:1 mixture of Cellstripper (Corning, catalog #25-056-CI) and TrypLE Express (1X) (Thermo Fisher Scientific, catalog #12604-013). Resulting suspension was filtered through a 40-µm filter, centrifuged at 350 g, and resuspended in wash buffer (PBS plus 0.5% bovine serum albumin). To isolate c-Kit+ cells, suspension was incubated with c-Kit–labeled beads (Miltenyi Biotec, catalog #130-091-332) and sorted according to the manufacturer's protocol. The c-Kit+ fraction was divided as such: half the population was suspended in CPC media and the other half was suspended in EPC media. To isolate an EPC population, at 1 week the c-Kit+ population plated in EPC media was further sorted using CD133–labeled beads and sorted according to the manufacturer's protocol (Miltenyi Biotec, catalog #130-097-049). All cells were cultured at 37°C in 5% CO2 incubator in their respective growth media. CPC and EPC were split 1:2 when they reached 60-70% confluency. MSC were split 1:2 when they reached 90% confluency. All cells used in this study were mid-passage (passages 5–10). The Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3' Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturers instructions in the Chromium Single Cell 3' Reagents Kits V2 User Guide.
identifiers.namespace: GEO
identifiers.id: GSM3928161