title: GSM7876270: Ctl1; Mus musculus; RNA-Seq
library_name: GSM7876270
library_construction_protocol: Whole heart samples were prepared using the single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) protocol published previously (Martin et. al 2023, doi: 10.1038/s41596-022-00752-0) with the following modifications. Briefly, 2 hearts from control and 2 hearts from KO mice were snap frozen in liquid nitrogen 2 weeks following MI. Nuclei were isolated from frozen tissues by grinding the frozen tissue with a mortar and pestle, lysing the powdered tissue in hypotonic lysis buffer B, and dissociating the tissue in a genlteMACS M tube run on the “Protein 001” program followed by 50 strokes in a Dounce homogenizer with a type A/loose pestle. Lysates were subsequently filtered through a 40μm filter and centrifuged at 500xg for 5 minutes at 4°C. The nuclear pellet was then resuspended in 0.3M SPBSTM and filtered through a 20μm filter. The nuclei were subsequently fixed in methanol/DSP for 5 minutes at room temperature. The fixed nuclei were subsequently rehydrated with 0.3M SPBSTM, spun down at 500xg for 5 minutes at 4°C, and resuspended in 0.3M SPBSTM. The fixed nuclei were then aliquoted, snap frozen, and stored at -80°C until sequencing. Libraries were prepared according to the single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) protocol published previously (Martin et. al 2023, doi: 10.1038/s41596-022-00752-0). Briefly, fixed nuclei were distributed to wells of a 96 well plate and underwent reverse transcription which introduced a first index. Nuclei were then pooled and re-allocated to wells of a 96 well plate for incorporation of a second index by ligation. Nuclei were then pooled and split a third time for introduction of a third index by second-strand synthesis. Finally, libraries were purified and sequenced.