Mus musculus
Protocols: The uterus was removed from 6-day pregnant dams at 9:00 am. 38 embryos were dissected to discard the extraembryonic portion and combined to ensure sufficient number of cells for analysis. The embryos were dissociated into single cells by incubating them in 200μl of TriplE Express for 8 minutes at 37°C with mild mixing every 2 minutes. To stop the TriplE Express, 1ml of ice-cold 10% FBS in PBS was added, and the cells were then filtered through a 40μM Flowmi cell strainer. Following centrifugation at 300g for 4 minutes, the supernatant was removed, and the cells were resuspended in 50μl of PBS containing 0.04% BSA. Cell counts and viability assessments were performed using trypan blue staining on a Countess II instrument (Invitrogen), confirming that over 95% of cells exhibited high sample quality.The isolation of cell nuclei was carried out following the low-cell input version of the 10X protocol (Protocol Link: https://assets.ctfassets.net/an68im79xiti/6t5iwATCRaHB4VWOJm2Vgc/bdfd23cdc1d0a321487c8b231a448103/CG000365_DemonstratedProtocol_NucleiIsolation_ATAC_GEX_Sequencing_RevE.pdf). In brief, the 50μl cell suspension was transferred to a 0.2ml PCR tube and centrifuged at 300g for 5 minutes. After removing the supernatant, the cells were resuspended in 50μl of ice-cold nuclear extraction (NE) buffer (containing 10mM Tris pH 7.5, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween, 1mM DTT, 1U/ul RNaseIn from Promega, 0.1% NP40, and 0.01% Digitonin) and incubated on ice for 4 minutes. Subsequently, 50μl of wash buffer (similar to NE buffer but without NP40 and digitonin) was added, and the nuclei were pelleted by centrifugation at 500g for 5 minutes at 4°C. Following removal of the supernatant, the nuclei underwent an additional wash with 50μl of diluted nuclei buffer (10x Genomics), were pelleted again, and eventually resuspended in 7ul of diluted nuclei buffer (10x Genomics). A 1μl sample was assessed for quality and nuclei counts using a Countess II instrument, revealing that more than 99% of nuclei stained positively for trypan blue and exhibited the expected morphology. The nuclei were then diluted. A total of 13750 nuclei were taken forward for 10x snATACseq library preparation. The uterus was removed from 6-day pregnant dams at 9:00 am. 38 embryos were dissected to discard the extraembryonic portion and combined to ensure sufficient number of cells for analysi as previously described: Sendra, M., Mañes, J., Domínguez, J. N. & Torres, M. Live Imaging of Early Cardiac Progenitors in the Mouse Embryo. J. Vis. Exp. 2022, e64273 (2022). NGS experiments were performed in the Genomics Unit of the CNIC. Nuclei were counted and their integrity was checked using the Countess III cell counter (Thermofisher). 13750 nuclei were taken for the transposition reaction and loaded into one port of a Chromium Next GEM Chip H (10x Genomics) with a target output of 7,000 nuclei. Single nuclei were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics).  Sn-ATAC-seq libraries were prepared using the Chromium Next GEM Single Cell ATAC Kit v1.1 (10x Genomics) following the manufacturer instructions and the library was amplified using a SureCycler 8800 thermal cycler (Agilent Technologies). The average size of the library was then calculated using a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and the concentration was determined using the Qubit fluorometer (Thermofisher).
strain
CD-1
isolate
not applicable
organism
Mus musculus
organism
Mus musculus
collection date
not collected
scientific_name
Mus musculus
common name
house mouse
organism part
Pre-streak embryo
developmental stage
E6.5
geographic location (country and/or sea)
not collected