GSM9230012: Multiome, e14.5 lung B6 control, ATAC; Mus musculus; ATAC-seq
Pooled E14.5 male mouse lungs of control and BMS493 treatment groups in A/J and B6 strains were used for the analysis. To collect enough single nuclei, embryonic lungs from four (B6) or six (A/J) mice were pooled and mechanically dissociated using the gentleMACS Octo Dissociator (Miltenyi Biotech, MD) with the “4C _nuclei_1” program. After dissociation, the sample was incubated on ice for 5 minutes, filtered through a 30 µm filter to remove larger particles, and centrifugated at 4°C 300xg for 5 minutes. The pellet was resuspended in 500 µl of cold resuspension buffer (1X PBS, 0.1% BSA, 0.2 U/µl RNase inhibitor) and then centrifugated at 4°C 300xg for 5 minutes. Eventually, the pellet was resuspended in 300 µl of cold resuspension buffer. To further purify and concentrate the nuclei suspension, the 300 µl of nuclei suspension was gently mixed with an equal volume of 50% OptiPrep (MilliporeSigma, MA) in a 2 ml tube and applied gently on top of a 29% OptiPrep (600 µl) cushion in another 2 ml tube followed by centrifugation at 4°C 10000xg for 30minutes. The OptiPrep supernatant was poured out in a single motion, and the pellet was resuspended in 0.1X lysis buffer (0.1X nuclei extraction buffer with 0.2 U/µl RNase inhibitor) for permeabilization according to the 10X Genomics nuclei permeabilization step (CG000375). Nuclei were then collected and resuspended in Diluted Nuclei Buffer (1X Nuclei Resuspension Buffer, 1mM DTT, and 1 U/µl RNase inhibitor). Single nuclei were counted with AO/PI staining using a DeNovix CellDrop FL (D.A.I. Scientific Equipment, IL). The integrity of isolated nuclei was inspected under a microscope at 100x magnification. The Multiome libraries were prepared according to the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC +Gene Expression user guide (CG000338). The library quality was evaluated using the Agilent 4200 TapeStation (Agilent Technologies, CA), and the library concentration was determined by the Qubit Fluorometer (Invitrogen, CA). The single-nuclei ATAC-seq (snATAC-seq) and single-nuclei RNA-seq (snRNA-seq) libraries were sequenced on the Illumina NextSeq 2000 using the 50-8-24-49 and 28-10-10-90 formats, respectively, aiming for 25000 snATAC-seq and 20000 snRNA-seq reads per cell to ensure data coverage.