Illumina HiSeq 4000 paired end sequencing; A gene regulatory network for neural induction
A gene regulatory network for neural induction
Fertilised chicken eggs (Henry Stewart & Co.Ltd, Norfolk UK) were incubated at 38°C. The embryos were removed from the egg, staged, and pinned ventral side up on a resin plate in Tyrode's saline. Embryos were dissected with a G-31 syringe needle. The endoderm and mesoderm were removed with a small volume of dispase (10mg/ml) before dissecting an ectodermal region spanning the neural plate and neural plate border (Figure 6A). Tissue samples were pooled from multiple embryos of the same stage prior to dissociation (HH4=~55 embryos; HH6=~45 embryos; HH8=10 embryos; HH9+=7 embryos). For cell dissociation, samples were incubated in FACSmax cell dissociation solution (Amsbio, T200100) with 30U/ml Papain (Sigma, P3125) at 37°C for 20 minutes. The tissue was pipetted 10 times every 5 minutes in order to facilitate mechanical dissociation. To stop enzymatic dissociation, an equal volume of resuspension solution (HBSS with 0.1% non-acetylated BSA (Invitrogen, 10743447), 1% HEPES, 1x non-essential amino acids (Thermo Fisher Scientific, 11140050) and 10M rock inhibitor (Stemcell Technologies, Y-27632)) was added before passing cells through a 20m filter (Miltenyi Biotech, 130-101-812). Samples were then centrifuged at 100rcf at 4°C for 5 minutes and resuspended in resuspension solution twice. Cells were then FAC sorted with 1ul 0.1mg/ml DAPI to remove dead cells and any remaining cell doublets. Immediately after FACS, samples were centrifuged and placed in 90% MeOH with 10% resuspension solution for storage.  Given the limited number of cells collected from individual dissection rounds and to obtain the required cell concentration for 10x, multiple rounds of dissections were carried out over successive days. Immediately prior to loading on the 10x Chromium controller, samples were pooled by stage and underwent three rounds of centrifugation at 1500rcf at 4°C for 10 minutes followed by rehydration with DPBS with 0.5% non-acetylated BSA and 0.5U/l RNAse inhibitor (Roche 3335399001). N/A cDNA synthesis and library preparation were carried out using the Chromium Single-Cell 3′ Reagent v3 Kit (10× Genomics, Pleasanton, CA) according to the manufacturer's protocol.