GSM5827716: SF7 WT; Astyanax mexicanus; RNA-Seq
Hearts were isolated and tissue digestion was based on a protocol previously described by Sander et al (2013). Hearts were digested in 500µL Perfusion Buffer. Perfusion Buffer was made fresh every digestion and consisted of: 1X PBS, 30mM Taurine (Sigma, cat. no. T8691), 5.5mM Glucose (Sigma, cat. no. G7528), 10mM 2,3-Butanedione Monoxime (Sig-ma, cat. no. B0753), 10mM HEPES (Sigma, cat. no. H3393), 12.5uM CaCl2 (Sigma, cat. no. C4901), 5mg/mL Collagenase II (Gibco, cat. no. 17101-015), 5mg/mL Collagenase IV (Gib-co, cat. no. 17104-019) and 30mg DNAse I (Sigma, cat. no. 10104159001). [CaCl2] concen-tration was kept at 12.5µM to ensure adequate cell survival of all cell types present in the heart. Ventricles were collected on ice, minced into small pieces, and digested in an Ep-pendorf tube with 500µL of digestion buffer. Samples were incubated at 32°C on an Ep-pendorf thermomixer at 800rpm. Supernatants were collected every 15 minutes, neutral-ised using 1% sheep serum (Sigma, cat. no. S3772) and samples were replenished with fresh digestion buffer until all tissue had been digested (approx. 1-2 hours). Dissociated cells were filtered through 100µm filters, spun down (300g for 5 mins at 4°C), counted and spun down again before resuspension. Cells were suspended at 2000 cells/µL in DMEM (Life Technologies, cat. no. 22320-022) plus 10% fetal bovine serum (ThermoFisher, cat.no. A3840001) before loading onto 10X Chromium Chip B (10x Genomics, cat. no. 1000073). Single Cell RNAseq libraries were generated using the 10X Chromium Next GEM Single Cell 3' v3.1 kit (10x Genomics, cat. no. 1000092) and sequenced using the Illumina NextSeq® 500/550 High Output Kit v2 (cat.no. FC-404-2005) in 4 runs.