title: GSM6205436: E8.75_rep1 [ATAC-Seq]; Mus musculus; ATAC-seq
library_construction_protocol: 10x Genomics Single Multiome 10x Genomics Single Multiome Following dissection, embryos from the same stages were pooled to give sufficient cell numbers. Embryos were dissociated into single-cells using 200μl of TriplE Express for 10 minutes at 37°C on a shaking incubator. 1ml of ice-cold 10% FBS in PBS was added to quench and cells were filtered using a 40μM Flowmi cell strainer. Following centrifugation at 300g for 5 minutes, the supernatant was discarded and cells were resuspended in 50μl of PBS containing 0.04% BSA. Cells were counted and viability assessed using trypan blue staining on a Countess II instrument (Invitrogen). >95% of cells were negative for trypan blue indicating high sample quality.     Nuclear isolation was carried out according to the low-cell input version of the 10X protocol for cell lines and PBMCs (https://assets.ctfassets.net/an68im79xiti/6t5iwATCRaHB4VWOJm2Vgc/bdfd23cdc1d0a321487c8b231a448103/CG000365_DemonstratedProtocol_NucleiIsolation_ATAC_GEX_Sequencing_RevB.pdf). Specifically, the 50μl cell suspension was transferred to a 0.2ml PCR tube and centrifuged at 300g for 5 minutes. After removing the supernatant, cells were resuspended in 50μl ice cold nuclear extraction (NE) buffer (10mM Tris pH 7.5, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween, 1mM DTT, 1U/ul RNaseIn (Promega), 0.1% NP40, 0.01% Digitonin) and incubated on ice for 4 minutes. 50μl of wash buffer (identical to NE buffer but lacking NP40 and digitonin) was added and nuclei were centrifuged at 500g for 5 minutes at 4°C. After removing the supernatant, nuclei were washed once in 50μl of diluted nuclei buffer (10x Genomics), span down and finally resuspended in 7ul of dilute nuclei buffer (10x Genomics). 1μl was used to assess quality using a microscope and count nuclei using a Countess II instrument. >99% of nuclei stained positive for trypan blue and the nuclei were found to have the expected morphology. Nuclei were diluted such that a maximum of 16,000 were taken forward for 10x Multiome library preparation. Libraries were prepared using the 10x Genomics Chromium Combined snRNA-seq and snATAC-seq using 10x Genomics Multiome