GSM7688782
Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tg(hmx CNEIII:cfos:GAL4-VP16,UAS:EGFP)SU41 background (generated as described above) were screened for fluorescence from 24 hpf onwards. Only EGFP-positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting (FACS) at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 µl of Buffer RLT (Qiagen RNeasy Micro Kit, 74004) plus 143 mMβ-mercaptoethanol. Sorted cells were stored at -80oC prior to RNA extraction.  Frozen FAC-sorted cell lysates were removed from storage at -80oC and thawed in a 37oC waterbath, before transferring to sterile microcentrifuge tubes. If necessary, sample volumes were completed to 250 µl with UltraPure DNase/RNase-Free distilled water (ThermoFisher Scientific, 10977035). 750 µl TRIzol LS Reagent (ThermoFisher Scientific, 10296028) was added to each 250 µl sample, before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes (which had been pre-centrifuged as per the manufacturer's instructions (ThermoFisher Scientific, A33248)), before incubating for 5 minutes at room temperature. 200 µl chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16,000 x g at 4oC, before transferring the RNA-containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase-free ethanol. Samples were inverted to mix thoroughly, and the supernatant immediately loaded to an RNEasy MinElute column (from the RNeasy Micro Kit, Qiagen, 74004), before centrifuging for 15 seconds at 10,000 rpm. Wash steps with RW1 buffer, RPE buffer and 80% RNase-free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 µl RNase-free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip (Agilent, 5067-1513) on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity (RIN) values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High-Sensitivity Assay Kit (ThermoFisher Scientific, Q32852) and a Qubit 3.0 fluorometer (ThermoFisher Scientific, Q33216). cDNA was synthesised using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634888), and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024). cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample (Illumina NextSeq 500/500 High Output Kit, v2.5, 75 cycles, 20024906).