title: GSM8600343: RNA JM22-59-2_E8.5_WT_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Embryos were dissected and, at later stages when yolk was present, also de-yolked, in ice-cold PBS with 1% fetal bovine serum. For E7.75 embryos, whole embryos were dissected and harvested for single-nucleus library generation. For E8.5 and E9 embryos, the head folds and posterior trunk were removed by microdissection prior to harvesting for library generation to enrich the relative capture of cardiac cell types. Rapid PCR genotyping was performed while dissected embryos remained on ice. Embryos selected for the multiome experiment were incubated in 200 μl TrypLE Select for 5 minutes at 37 °C, triturated gently by pipetting up and down, and then incubated an additional 3 minutes at 37 °C. The dissociated cell suspension was quenched with 600 μl of PBS with 1% FBS, singularized by passage through the 35 μm mesh of a 5 mL Falcon™ Round-Bottom Polystyrene Test Tube with Cell Strainer Snap Cap, pelleted by centrifugation at 300 g for 5 minutes at 4 °C, and resuspended in 50 μl of PBS with 0.04% bovine serum albumin (BSA). At this stage, the manufacturer's protocol for Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing, Appendix, Low Cell Input Nuclei Isolation (10x Genomics, CG000365 Rev B) was followed exactly to prepare nuclei for library generation. Library construction was performed according to manufacturer's protocols (10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide, CG000338 Rev B). Following dissection and rapid genotyping, embryos selected for the multiome experiment were processed directly into the transposition reaction and then captured in 10x Genomics GEMs via the Chromium Controller. GEMs were stored at -80 °C for up to 4 weeks, allowing for collection of replicate embryos of each genotype at each development stage. All subsequent library preparation steps were performed together for all embryos of a given developmental stage to reduce the likelihood of technical batch artifacts. A targeted maximum recovery of 10,000 cells per sample were loaded onto the 10x Genomics Chromium controller instrument. Each sample's snRNA-seq library was indexed with a unique sample identifier with the Dual Index Kit TT Set A (10x Genomics). Final libraries were quality-controlled using an Agilent Bioanlyzer instrument with Agilent High Sensitivity DNA Kit. The DNA concentration of each library was measured using KAPA Library Quantification qPCR Kit.