title: GSM9558101: 090622ICN_Near_E14_5; Mus musculus; RNA-Seq
library_construction_protocol: For scRNA-seq of OINS cells, the heart, lungs, pancreas and intestines were dissected from Wnt1-Cre;lox-tdTomato, Phox2b-Flpo;frt-tdTomato, or Baf53b-Cre;lox-L10GFP mice as indicated at E12.5, E14.5, E16.5, E18.5 and P56 in ice-cold Leibovitz's L-15 Medium (Gibco). For the hearts and lungs, fluorescently labelled OINS regions were isolated by microdissection under a Leica M205FCA fluorescence stereomicroscope. For experiments comparing heart-near and heart-far regions, atrial areas containing Wnt1-Cre;lox-L10GFP labelled ICNS cells were dissected to separate from the atrial areas lacking ICNS. Dissected tissues were cut into 1-3 mm2 pieces and incubated in 0.25% trypsin-EDTA (Gibco) at 37 ˚C for 10 minutes (E12.5, E14.5), 20 minutes (E16.5, E18.5), or 25 minutes (P56) with shaking. After washing with L-15 media containing 10% fetal bovine serum (L15/FBS), tissues were incubated in 2 mg/mL Collagenase A (Sigma-Aldrich) and 2 mg/mL of Dispase II (Sigma-Aldrich) in 1 x HBSS at 37°C for 30 minutes (E12.5, E14.5) or 40 minutes (E16.5, E18.5, P56) with shaking. After washing with L15/FBS, cells were separated into single cells by trituration using three fire-polished Pasteur pipettes of decreasing diameter. Mature ICNS-neurons isolated from Baf53b-Cre;lox-L10GFP hearts at E18.5 and P56 were further purified using 30% and 60% Percoll density gradients. The cell suspension was filtered through a 40 μm cell strainer (Corning). All centrifugation steps were performed at 200 x g at 4°C. After the final centrifugation, the cell pellet was resuspended in 1x HBSS containing 0.04% BSA. Fluorescently labelled cells were sorted on a BD FACS Aria cell sorter at Yale Flow Cytometry Facility and collected in ice-cold 1x HBSS containing 0.04% BSA.  For co-culture experiments, after 4-5 days of culture, media were removed and cells were incubated with 2 mg/mL Collagenase A (Sigma-Aldrich) and 2 mg/mL of Dispase II (Sigma-Aldrich) solutions at 37 ˚C for 10 minutes. The digested cells were collected into ice-cold 10% FBS L-15 media (L15/FBS). This digestion was repeated 3-6 times until the coating layer was completely dissociated. Cells pooled from multiple wells were centrifuged at 200 x g at 4 ˚C for 10 minutes, washed three times with L15/FBS, and treated with Corning Cell Recovery solution (#354253) on ice for 20 minutes to dissolve residual Matrigel. After two additional washes with L15/FBS, cells were gently triturated with a Pasteur pipet, filtered through a 40 μm cell strainer, washed three times, and resuspended in 1x HBSS, 0.04% BSA for scRNA-seq using the 10x Genomics platform. Single-cell libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Reagent Kit v3 chemistry according to the manufacturer's instructions and sequenced using an Illumina NovaSeq 6000 S4 sequencer at 300 million reads to achieve a sequencing depth of 20,000–300,000 reads per cell.