title: 10x single cell RNAseq of chick ectoderm from stages HH5 to ss4
design: 10x single cell RNAseq of chick ectoderm from stages HH5 to ss4
library_construction_protocol: Fertilised chicken eggs were incubated at 38°C for 24-32 hours depending upon the developmental stage collected. Embryos were pinned and had the endoderm and mesoderm removed using dispase (10mg/ml) before dissecting the ectoderm. Multiple embryos from the same stage were pooled prior to dissociation. Samples were then dissociated in 250ul pre-heated FACSmax cell dissociation solution (Amsbio, T200100) with papain (30U/ml; Sigma, P3125) for 20 minutes at 37°C. Samples were gently pipetted every 5 minutes to facilitate dissociation. After dissociation, 250ul resuspension solution was added to prevent any further dissociation (HBSS; non-acetylated BSA (1mg/ml; Invitrogen, 10743447); Hepes (0.01M); non-essential amino acids (1x; Thermo Fisher Scientific, 11140050); rock inhibitor (10M; Stemcell Technologies, Y-27632). Cells were passed through a 20um cell strainer (﻿Miltenyi Biotech, 130-101-812) to remove any debris, before pelleting and re-suspending in 500ul resuspension buffer. To remove any dead or dying cells as well as any remaining doublets, 1ul 0.1mg/ml DAPI was added to the cell suspension before FAC sorting. Given the low number of cells obtained from each embryo, samples for each stage were collected over multiple days and stored in 90% MeOH with 10% resuspension solution after FAC sorting. Prior to sequencing, samples from the same stage were pooled and resuspended in DPBS with 0.5% non-acetylated BSA and 0.5U/ul RNAse inhibitor (Roche 3335399001). Samples were loaded onto the Chromium controller along with 10x single cell 3' kit reagents. cDNA synthesis and library preparation was carried out at the Francis Crick institute using the Chromium Single-Cell 3' v3 Kit in accordance with the manufacturers protocol. Libraries were checked on an Agilent TapeStation.