title: GSM6423676: H2CKO; Mus musculus; RNA-Seq
library_construction_protocol: E11.5 embryos were dissected in cold PBS and placed in PBS with 1% FBS solution on ice until dissociation (approximately 3 hours). Yolk-sac DNA was extracted (QuickExtract DNA Extraction Solution, Epicentre) and used for genotyping to distinguish heterozygous and homozygous Hand2 conditional allele. The RosamTmG allele fluorescence was used to determine Nfatc1cre status. Dissected cardiac tissue was incubated in 750 μl TrypLE (ThermoFisher) for 5 min at 370C, triturated with a 200-μl wide-bore pipette tip. The cell suspension was quenched with 750 μl DMEM with 10% FBS. Cells were filtered through a 30-μm cell strainer (MACS SmartStrainer), centrifuged at 300g for 5 min, and washed once with 750 μl PBS with 0.5% BSA. Cells were resuspended in 30 μl PBS with 0.5% BSA (10x Genomics). Single-cell droplet libraries were generated using the Chromium NextGEM Single Cell 3' Reagent Kits User Guide, CG000204 Rev D (10X Genomics, Inc). according to the manufacturer's instructions. Briefly, each clean single cell suspension was counted with hemocytometer under microscope for cell number and cell viability. Only single cell suspensions with a viability of >90% and minimal cell debris and aggregation were used for further processing. The resulting library was sequenced in a custom program for 28b plus 91b paired-end sequencing on Illumina NovaSeq 6000. About 50K reads per cell were generated and 91% of the sequencing reads reached Q30 (99.9% base call accuracy). A Phred quality score (Q score) was used to measure the quality of sequencing.