GSM5221132: Control_E12.5OFT_scRNAseq; Mus musculus; RNA-Seq
E12.5 cardiac OFTs (both Notch1+/- and wildtype) were microdissected from five mouse embryos and pooled for single-cell RNA sequencing. To prepare single cells suspensions, pooled OFT tissues were incubated with 1 mg/ml Collagenase II (Worthington Biochemical Corporation# LS004176) for 15 mins at 37°C with occasional stirring at every 5 minutes for complete dissociation. The digestion reaction was quenched immediately with 1 ml. DMEM supplemented with 10% FBS and pelleted down at 1000 rpm for 5 mins at 4°C. Pelleted cells were washed with cold PBS and resuspended in 0.04% BSA in PBS at concentration 9.74 X 105 cells/ml with 83% viability (8.03 X 105 live cells/ml). A single-cell droplet library of the pooled Notch1+/-  / wildtype OFTs samples was generated using the 10xGenomics Chromium controller from this suspension according to the manufacturer's instructions. The quality and integrity of the cDNA library was quantitated using the High Sensitivity D5000 and D1000 ScreenTape (Agilent# 5067-5592 and 5067-5584) on the Agilent-2200 TapeStation. The library was sequenced (150bp paired-end) using the Illumina HiSeq 4000 platform at the Steve and Cindy Rasmussen Institute for Genomic Medicine at Nationwide Children's Hospital. A total of 337,081,925 paired end reads and a total of 2,116 cells were detected for the pooled sample. Thus, on average, the transcriptome of each cell was evaluated using 159,301 reads.