title: GSM6940808: 7S,  prefix: RIT735A3, replicate 1, scRNAseq; Gallus gallus; RNA-Seq
library_name: GSM6940808
library_construction_protocol: Fertilized chicken eggs obtained from Henry Stewart & Co. Ltd were incubated at 37°C with ~40% humidity for 29 to 45 hours to yield a minimum of 4 embryos with specific somite numbers: 4, 7, 10 and 13somites. All embryos were dissected to keep solely the tissue caudal to the third somite (inclusive). A single-cell suspension was obtained by incubating the dissected embryos at 37°C in a dissociation solution consisting of accutase (Stemcell Technologies) with 3U/mg Papain (Sigma-Aldrich, 10108014001) and 1mg/mL of Collagenase 4 (Gibco, 17104019) for 20 min. Half-way through incubation and at the end, the embryos were mechanical dissociated with a P1000 pipet. After dissociation 200uL of resuspension buffer (DMEM/F12 with 1% BSA) was added. The cell suspension was then spined for 4min at 0.6xg, resuspended in 250µL of resuspension buffer and filtered through a 40µm Flowmi cell strainer (cat. no. 136800040) and twice through a pre-wet 20µm pluriSelect strainer (43-10020-60). The yield and cell viability for the 4S, 7S, 10S and 13S samples were 640, 500, 1300 and 1600 cells/µL with a viability of 88%, 93%, 92% and 95% respectively. A single-cell suspension was loaded independently for each sample onto the channels of Chromium Chip G for use in the 10x Chromium Controller (PN-1000120) with the goal of obtaining 10000 cells. The cells were partitioned into nanolitre scale gel beads in emulsions (GEMs) and lysed using the 10x Genomics Single Cell 3′ Chip V3.1 GEM, Library and Gel Bead Kit (PN-1000121). cDNA synthesis and library construction were performed as per the manufacturer's protocol for the Chromium Single-Cell 3′ mRNA V3.1 protocol. cDNA amplification involved 12 PCR cycles. Libraries for the samples were multiplexed so that the number of reads matched one lane per sample and sequenced on an Illumina HiSeq4000 using 100 bp paired-end runs.