GSM8147316: RARa403 E4 quail NTs; Coturnix japonica; RNA-Seq
At E4, ten NTs per group (with associated mesoderm) were mechanically microdissected in Phosphate-buffered saline (PBS) supplemented with Ca2+/ Mg2+ and 5% fetal calf serum (FCS). NTs were incubated for 15 min at 37oC in 1.5 ml Accutase (Sigma Israel, A6964) containing 30 ul Papain (Sigma Israel, P3125) and 300 ng DNaseI (Sigma Israel, DN25) and dissociated mechanically using sequentially 1000 and 200 ul pipettes. Cells were filtered through a 40-micron filter and resuspended in PBS containing 2% FCS. Samples were sorted using a BD FACSAria III sorter (BD Biosciences UK). Single cells were isolated by sequentially gating cells according to their SSC-A vs. FSC-A and FSC-H vs FSC-W profiles, following standard flow cytometry practices. Dead or damaged cells were excluded following Propidium Iodide uptake. Citrine fluorescence was detected using a 488 nm laser;  age-matched NT cells from untransfected embryos served as negative control to determine the fluorescence gate. Sorted fluorescent cells were loaded into the 10X Genomics Chromium Next controller.  Libraries were prepared following manufacturer's instructions (GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1, 10× Genomics, CA, USA). Approximately 10.000 cells were loaded per sample. Sequencing was performed using the Illumina Nextseq500 platform (Illumina Inc, San Diego, CA,USA) with the following sequencing conditions: 28 bp (Read1) and 54 bp (Read2).