GSM8833513: multiome, E15.5 back skin from 3 Prmt5;cKO embryos (pooled), ATAC, pool 3; Mus musculus; ATAC-seq
For single cell multiome-sequencing experiments, embryos were collected at E15.5 from multiple litters. Yolk-sac tissue was lysed quickly using the Extract-N-Amp Tissue PCR Kit (Sigma-Aldrich) and embryos quickly genotyped using Phusion High-Fidelity DNA Polymerase (New England Biolabs) and shortened PCR cycling parameters. During genotyping, the back skin from all E15.5 mouse embryos was dissected and immediately transferred to ice cold DEPC-PBS (Millipore Sigma). Samples were incubated in 0.25% Trypsin (ThermoFisher) for 30-45 mins at 37°C with frequent pipette mixing. Once cells reached a single cell suspension, the reaction was quenched with 10% FBS (ThermoFisher), passed through a 40 µM cell strainer (Flomi Cell Strainers, SP Bel-Art), and triplicates of each genotype (CRE-negative controls or Prmt5 cKOs, based on quick-genotyping results) pooled. 500,000 cells from each pooled sample were incubated with lysis buffer (10mM Tris HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40) for 5 mins on ice, nuclei extracted and quantified using trypan blue. scRNA-Seq and scATAC-Seq sequencing libraries were prepared by the University of Iowa's Iowa Institute of Human Genetics Genomics Division Laboratory. Briefly, 10,000 nuclei were targeted for bulk transposition before individual nuclei were encapsulated in oil droplets along with the 10X GEM code beads in the Chip J cartridge (P/N: 1000234) using the 10X Genomics Single Cell iX Chromium Controller. Generation of gel beads in emulsion (GEMs), barcoding, pre-amplification PCR, and ATAC library construction were all performed as recommended by the manufacturer (10X Genomics, Chromium Next GEM Single Cell Multiome + ATAC Reagent Kit, Rev F, P/N:1000283). The gene expression libraries were pooled and sequenced on a NovaSeq 6000 to give at least 20,000 reads per nuclei. The ATAC-Seq libraries were pooled and sequenced on a NovaSeq 6000 to give at least 25,000 reads per nuclei.