title: GSM9012231: 4wpf mutant; Danio rerio; RNA-Seq
library_construction_protocol: Extraction was performed as described by Mazzolini et al, 2018, with modifications. Eyecups were dissected and mashed in a dounce homogenizer containing 15 mM HEPES and 25 mM D-Glucose in 1x HBSS (media A). The samples were washed, strained and resuspended in 22% density gradient medium (100% is prepared as 9:1 Percoll density gradient medium : 10x HBSS) overlaid with 1x DPBS. Then they were centrifuged, washed in media A + 2% normal goat serum, and finally resuspended in 1x DPBS + 2% normal goat serum. Once resuspended, Sytox blue dead cell stain (#S34857) was added at a dilution of 1:1000. Cell sorting was performed immediately on the BD FACSAria II and FACSAria III. Sorting parameters were standardized using fish bearing no fluorescence-expressing transgene as a control, and mfap4:tdTomato-CAAX fish as the test sample at the relevant ages (5 days or 4 weeks post fertilization). Microglia/macrophages were sorted by gating on FSC/SSC scatter, Sytox blue dead stain, and tdTomato intensity. FACS-isolated cells were loaded onto Chromium Next GEM Chips (10x Genomics). Libraries for sequencing were then prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3 or Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) (10x Genomics), following the manufacturer's protocols. 12 cycles were used for the cDNA amplification PCR and 14 cycles were used for the indexing PCR.