GSM7688795
Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP-positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked, dissected and dissociated as described in GSE145916, with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord, and posteriorly, immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System (Worthington Biochemical Corporation, LK003150), trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately after homogenising the digested tissue mixture with a sterile p200 tip, we passed each sample through a 40 µm Flowmi cell strainer (Merck, BAH136800040) into a sterile microcentrifuge tube. After Papain inactivation, samples were resuspended in 1 ml Leibovitz's L-15 medium (ThermoFisher Scientific, 21083027) + 0.5% FBS and stored on ice. Immediately before FACS, DAPI (Merck, D9542) and Draq5 (BioLegend, 424101) were added at a final concentration of 5 µg/ml and 5 µM respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al., (2008) with the following modifications. Ice-cold samples were filtered through 35 µm mesh strainers in to 5 ml round-bottomed polystyrene tubes (Corning Falcon, 352235). All FAC-sorting and collection steps were performed at +4oC, using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates (forward scatter height x forward scatter width, followed by side scatter height x side scatter width) were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI-negative (sick cells are DAPI-permeant and excluded) and Draq-5 positive (only healthy nuclei are Draq-5 permeant) cells.  EGFP-positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” (https://www.10xgenomics.com). EGFP-positive cells were sorted directly in to 5 ml round-bottomed tubes containing 3.5 mls of freshly made, pre-chilled, 90% methanol (for HPLC, >99%, Merck, 34860)/10% Dulbecco's Phosphate-Buffered Saline (DPBS, No calcium, No magnesium, Merck, D8537) fixative. A tube of EGFP-negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP-negative control tube using Trypan Blue (ThermoFisher Scientific, 15250061) and a hemocytometer. Samples with intact, fully fixed cells, containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single-cell capture with the 10x Genomics Chromium system (please see below).  To rehydrate our fixed EGFP-positive 48 hpf evx1i232;evx2sa140;Tg(evx1:EGFP)SU2 cells, we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing-bucket centrifuge. Next, we carefully removed the majority of the supernatant with a sterile p1000 tip, until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made, pre-chilled Rehydration Buffer (1x Dulbecco's Phosphate-Buffered Saline, no calcium, no magnesium (Merck, D8537), 1.0% UltraPure BSA (ThermoFisher Scientific, AM2616), 0.5 u/µl Roche Protector RNase Inhibitor (Merck, 3335402001)) and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. After the second Rehydration step, we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30-40 µl of supernatant. Using a sterile p200 tip, we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio-Rad TC20 automated cell counter (Bio-Rad, 1450102). We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” (https://www.10xgenomics.com), we too recovered approximately 50% of the sorted cells after rehydration. We isolated single cells using a 10x Genomics Chromium system, aiming for capture of 10,000 cells per well (Chromium Next GEM Chip G Single Cell Kit, 1000127). We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit (v3.1, 10x Genomics, 1000128) and sequenced them on an Illumina NextSeq500 to a depth of at least 50,000 reads per cell (Illumina NextSeq 500/500 High Output Kit, v2.5, 150 cycles, 20024907).