GSM8389636: Chicken_Day2_Rep2_scRNAseq; Gallus gallus; RNA-Seq
Isolation of birthdated cells for scRNAseq was performed as previously described(28). Briefly, chick and mouse embryos were birthdated at early neurogenic timepoints (E4 or E12, respectively) and sacrificed either two or eleven days after EdU injection. P3 postnatal mouse pups and E15 chick embryos were anesthetized by hypothermia after immersion of either pup or the chick egg in ice. Brains were extracted in ice-cold HBSS. Then, the pallial region was microdissected under a stereomicroscope, collected in ice-cold HBSS, pooled and cut into small pieces. Tissue was dissociated in a species and stage specific manner, according to Supplementary Table S3.Following enzymatic digestion, chick (both E6 and E15) and mouse P3 tissue was manually homogenized by carefully pipetting and tissue clogs were removed by filtering the cell suspension through a 40 μm nylon strainer to a 15 ml Falcon tube containing 4 mlof 25% fetal bovine serum (FBS) in HBSS. After mouse E14 tissue digestion, FBS was added to the mix, cells were manually dissociated by pipetting and tissue clogs were removed by filtering the cell suspension though a 40 μm nylon strainer. Dissociated cells were then centrifuged at 200g for 10 min at room temperature (RT) and EdU molecule was detected in the pelleted cells. Following reaction, cells were washed thrice by centrifugation at 200g for 10 min and suspended in 1 ml of HBSS then passed on a 50 μm cell strainer. PI-/EdU+ cells, gated to include only the top 10% brightest cells, were finally FAC-sorted: for the E6 chick E6 a Synergy 3200 2L sorter (Sony) was used; for the E15 chick sample a FACSAria (BD Biosciences) was used; and for both mice samples a FACS Jazz (BD Biosciences) was used. Cells were processed for single cell GEM formation following the standard Chromium Single Cell 3' v3.1. Briefly cells were placed at the Chromium chip with the beads and reagents for the RNA capture and cDNA amplification. After the emulsion were obtained, the cDNA was amplificated in a thermocycler. Post amplificated cDNA, now marked with individual cell barcode, were fragmented, ligated the sequencing adapters and amplified. After that, the PCR product were purified and quantified. Single cell library was prepared using “Chromium Next GEM Chip G Single Cell Kit”, 16 rxns PN-1000127, “Chromium Next GEM Single Cell 3ʹ Kit v3.1”, 4 rxns PN-1000269 and “Dual Index Kit TT Set A”, 96 rxns PN-1000215, following “Chromium Next GEM Single Cell 3' Reagent Kits v3.1(Dual Index) User Guide” (Document number CG000315). Resulting libraries were sequenced on a Novaseq 6000 for an approximated 50.000 reads per cell.