title: GSM9054345: M6alone_1_M6alone_2, CITE-seq; Mus musculus; OTHER
library_construction_protocol: Single cell libraries were generated from non-myocytes acquired by differential adherence to plastic following langendorff digestion of mice of the following genotype: wildtype Cre controls (Myh6MerCreMer ;Wt1CreERT2;Pkp2+/+), Pkp2myoKO (Myh6MerCreMer ; Pkp2fl/fl), and Pkp2myo/epi-KO (Wt1CreERT2;Pkp2fl/fl). TotalSeq-A antibodies (BioLegend, San Diego, CA) were added to the flow-sort antibody panel during cell labeling and following sorting, cell suspensions were loaded on a Chromium controller (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per manufacturer's recommendations. Briefly, the beads were dissolved, cells were lysed, and GEM reverse transcription (GEM-RT) was performed to produce barcoded, full-length cDNA. After incubation, GEMs were broken and the pooled post-GEM-RT reaction mixtures were recovered and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher Scientific, Waltham, MA). cDNA was amplified by PCR and fractionated by SPRI bead size-selection to separate mRNA-derived cDNA and CITEseq amplicons. cDNA amplicon size was optimized by enzymatic fragmentation and size selection. Indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. In parallel, CITE-seq library construction was performed following SPRI bead purification using Q5 Hot Start HiFi Master Mix (New England Biolabs, Ipswich, MA), SI PCR primer (IDT, Coralville, IA), and indexed TruSeq Small RNA PCR primers (Illumina, San Diego, CA) as specified in Stoeckius et al. Amplified CITE-seq libraries were purified using AMPure XP (Beckman Coulter, Indianapolis, IN) beads and quantified by Qubit dsDNA assay (ThermoFisher) and Bioanalyzer HSDNA (Agilent, Santa Clara, CA) analysis.  CITE-seq libraries were pooled with 10x Genomics gene expression libraries for sequencing on Illumina's NovaSeq 6000.