GSM9207598: scRNA_E115_tg_rep2; Mus musculus; RNA-Seq
Dissociation of DS1 and DS2U at hiPSC stage (Day 0) was achieved using Accutase, whereas Day 2, Day 4, Day 6, Day 8, Day 10, Day 20 cardiomyocytes were treated with 0.25% Trypsin. Preparation of CROP-seq samples was similar at both hiPSC and Day 20 cardiomyocyte stages and CROP-seq amplicon library preparation was prepared as previously described46,57. Briefly, after the cDNA isolation step of 10X scRNA-seq library preparation, 10 ng of cDNA was separated from the remaining library and subjected to three rounds of nested PCR steps using primers listed below. Amplicon library purity was assessed by bioanalyzer HS DNA kit and purified using SPRI clean up beads. Amplicon libraries were pooled and sequenced on Illumina NextSeq 500 (Illumina, software 4.0.2) using similar cycling conditions as the endogenous transcriptome libraries mentioned above. Embryonic day 11.5 (E11.5) mouse embryos were harvested from timed pregnant females, and genotyping was conducted using yolk sac DNA to identify wild-type, Dp1Tyb, Hmgn1+/−, and Dp1Tyb; Hmgn1+/− genotypes. For each embryo, the heart was micro-dissected in ice-cold phosphate-buffered saline (PBS) under a stereomicroscope. Individual hearts were enzymatically dissociated using TrypLE Express (Thermo Fisher Scientific), with samples incubated at 37°C in a water bath for 10 minutes. Gentle trituration was performed using a 200 µL pipette at both the 5-minute and 10-minute time points to promote dissociation. The enzymatic reaction was quenched by adding ice-cold PBS containing 1% fetal bovine serum (FBS). Each sample was then passed through a 40 µm cell strainer to obtain a single-cell suspension, followed by centrifugation at 400 × g for 10 minutes at 4°C. The cell pellet was washed once with 1% FBS in PBS and resuspended in 50 µL of the same buffer. Cell viability and concentration were assessed using trypan blue exclusion. Samples with greater than 85% viability were used for library preparation. Approximately 10,000 cells per sample were loaded onto the 10x Genomics Chromium platform using the 3' v4 chemistry. 10X Genomics v3 and v4 3' scRNAseq