A dual genetic constraint underlies the conservation of early brains in vertebrates
A dual genetic constraint underlies the conservation of early brains in vertebrates
Embryos were collected in RNAase free conditions and dissected to extract the brain (neural tube). Microdissection tweezers were employed to dissect out the neural tube. Starting by removing the optic vesicle and pulling the surrounding mesenchimal tissue surrounding. Similarly, the pharingeal arches, nasal placode and otic vesicle were also removed. Then, the neural tube was split in prosencephalon, mesencephalon and rhombencephalon (up to the optic vesicle) for further enzymatic desegregation into cells. After further mechanical desegregation, cells were collected, incubated with Tn5 transposase for ATAC or directly inputted into 10X Chromium machine for cell isolation in scRNA-seq. Chick: Fertilized chick eggs (Gallus gallus) were purchased from Granja Santa Isabel and incubated at 37.5 °C in humidified atmosphere until required developmental stage. The day when eggs were incubated was considered embryonic day (E)0.  Mouse: Adult C57BL/6 mice (Mus musculus) were obtained from a mice breeding colony at Achucarro Basque Center for Neuroscience (Spain). They were housed in a 12/12-hour light/dark cycle (8 AM, lights on) and provided with ad libitum food and water. T The day when the vaginal plug was detected was referred to as embryonic day 0.5 (E0.5) -to accommodate to La Manno et al (2021) dating. Gecko: Fertilized ground Madagascar gecko eggs (Paroedura picta) were obtained from a breeding colony at Achucarro Basque Center for Neuroscience. They were housed in a 12/12-hour light/dark cycle (8 AM, lights on, 27ºC/ 8 PM light off, 22ºC) and provided with ad libitum food (live crickets) and water. Gecko eggs were incubated at 28 °C in a low humidified atmosphere until required developmental stage. The day when eggs were incubated was considered E0.  No treatment was applied as all samples were obtained on wild type conditions. No treatment was applied as all samples were obtained on wild type conditions. No treatment was applied as all samples were obtained on wild type conditions. In both, snATAC-seq and scRNA-seq, barcoded fragments were generated for each bead-cell (Gel Beads-in-emulsion (GEMs)). For this step, oligonucleotides containing (i) an Illumina P5 sequence, (ii) a 16 nt 10x Barcode and (iii) a Read 1 (Read 1N) sequence were released in each GEM and mixed with DNA fragments and Master Mix. After incubation and amplification, the GEMs were broken and pooled. Next, P7 and a sample index were added during library construction via PCR.   In both, snATAC-seq and scRNA-seq, barcoded fragments were generated for each bead-cell (Gel Beads-in-emulsion (GEMs)). For this step, oligonucleotides containing (i) an Illumina P5 sequence, (ii) a 16 nt 10x Barcode and (iii) a Read 1 (Read 1N) sequence were released in each GEM and mixed with DNA fragments and Master Mix. After incubation and amplification, the GEMs were broken and pooled. Next, P7 and a sample index were added during library construction via PCR. 