GSM5585064: chd8+/+ 4dpf eNCC #3; Danio rerio; RNA-Seq
chd8+/+ and chd8 sa19827/sa19827 males were crossed with Tg2(phox2bb:EGFP) females, and the eggs were incubated at 28.5 °C.  At 4 days post-fertilization (dpf), the larvae were euthanized in 2 mg/ml tricaine diluted in RPMI and the heads of the larvae were discarded. The rest of the larval bodies were collected in a 2 ml Eppendorf tube, all RPMI was removed and replaced with 1 ml of Trypsin-EDTA 1X (Sigma, ref 59417C-100ML).  The digestion was stopped after 10 minutes by adding 50 µl of inactivated fetal calf serum. The tubes were centrifuged at 2000 g, during 2 minutes at room temperature, the supernatant was removed and 100 µl of FACS Max medium were added (AMSBIO, ref T200100).  The larval bodies were then placed on a cell filter (diameter 40 µm, Dutscher, ref 141378C), previously moistened with 100 µL of FACS Max medium, and the cells were filtered, using a 1 ml syringe plunger. The filter was rinsed with 400 µl of FACS Max medium, the cells were collected and placed in a 1.5 ml Eppendorf tube. The GFP-positive cells were immediately sorted, using an ARIA Fusion cell sorter and an excitation wavelength of 488 nm.  We stored the GFP-positive cells at -80 °C, in 10 µl of PBS-RNAsine 1 U/µl. Full length cDNA were generated directly from 950 to 1300 cells frozen in PBS buffer containing RNAse inhibitor using Clontech SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 12 cycles of PCR for cDNA amplification by Seq-Amp polymerase. 600 pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, CA) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoresis.