GSM5782165: Lower jaw of oral lamina adjacent to replacement teeth; Maylandia zebra; RNA-Seq
Nuclei preparation methods were based on the 10x Genomics Demonstrated Protocol for lysis and washing of single nuclei. To this protocol the following modifications were made: 1x HBSS was used in place of HEB; tissues were placed in lysis buffer for 10 minutes (to account for a smaller tissue mass) and then homogenized with disposable pellet pestles (Sigma-Aldrich Z359947) before halting lysis with HBSS; the tissue was suspended in 300 µL of wash buffer and passed through a 30μm MACS SmartStrainer once before proceeding to FACS. Filtered samples were stained with DAPI at a concentration of 0.1 μL DAPI per 100 μL of suspension and sorted using the BD FACSAria Fusion. Gates were set conservatively to collect DAPI+ singlet nuclei (Supplemental Figure 2) and nuclei were sorted into 200 µL of wash buffer. Total volume in each collection tube was recorded, collected nuclei were centrifuged at 300 rcf, 4°C for 10 minutes and supernatant was removed, leaving nuclei suspended in 50 µL of wash buffer. Event counts registered during sorting were used to estimate final cell suspension concentrations: from upper jaw soft tissue, 20668 nuclei were collected for a final concentration of 413 nuclei/µL; from lower jaw soft tissue, 20181 nuclei were collected for a final concentration of 403 nuclei/µL; from regenerating teeth 14700 nuclei were collected for a final concentration of 294 nuclei/µL. Libraries were prepared for sequencing by the Genome Analysis Core at Georgia Tech using the 10x Chromium V3 Single Cell Gene Expression Solution protocol and reagent kit (10x Genomics 1000092) with a targeted cell recovery of 3,000 nuclei per sample. NextSeq 500 (Illumina SY-415-1001)