GSM6752787
Single-cell suspensions were prepared from pooled paired secondary palatal shelves of three embryos at E12.5, two embryos at E13.5, and one embryo at E14.0 and E14.5. The micro-dissected palatal shelves were treated with 0.25% trypsin and 0.05% EDTA (150 μL) for 5 min at 37 °C with gentle agitation (300 rpm). The dissociated cell mixtures were then suspended with 300 μL Dulbecco's Modified Eagle's Medium (DMEM, Millipore Sigma) supplemented with heat-inactivated 10% fetal bovine serum (FBS).The cells were centrifuged at 500 g for 5 min at 4 °C and the cell pellets were resuspended and incubated with chilled 300 μL of 0.1x Lysis Buffer [10 mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1mM DTT, 1U/μL RNase inhibitor, 0.01% Tween20, 0.01% Nonidet P40 Substitute, and 0.001 % Digitonin] for 3 min on ice, followed by stopped with chilled 300 μL Wash Buffer [10 mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1mM DTT, 1U/μL RNAase inhibitor, and 0.1% Tween20]. The cells were then collected by centrifuge at 500 g for 5 min at 4 °C, rinsed with 200 uL Wash Buffer twice, and resuspended in Diluted Nuclei Buffer [1x Nuclei Buffer, 1 mM DTT, and 1U/μL RNase inhibitor]. Isolated single-cell nuclei were filtered using a cell strainer (40 μm pore size) and inspected under a microscope to ensure they were successfully dissociated into single cell level for subsequent sequencing. The single cell libraries were constructed by following the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (CG000338). Briefly, nuclei suspensions were incubated with a transposase, which fragmented the DNA in open regions of the chromatin and added the adapter sequences to the ends of the DNA fragments. The transposed nuclei were loaded onto Chromium Next GEM Chip J (PN-1000234, 10xGenomics, Pleasanton, CA) with partitioning oil and barcoded single-cell gel beads, followed by PCR amplification. The ATAC library and the gene expression library were then prepared separately. The quality of the libraries was examined using Agilent High Sensitive DNA Kit (#5067-4626) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). The library concentrations were determined by qPCR using Collibri Library Quantification kit (#A38524500, Thermo Fisher Scientific) on a QuantStudio3 (ThermoFisher Scientific). We then pooled the libraries evenly and performed the paired-end sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using High Output Kit v2.5 (#20024907, Illumina, Inc., USA). Multiome (snRNA-seq and snATAC-seq)