title: GSM8070855: KO_GC111891_RNA; Mus musculus; RNA-Seq
library_construction_protocol: Seven-day-old pups were decapitated and hearts were immediately removed and placed in PBS, all done on ice. The atria, part of the base and the entire right ventricle were removed, and the remaining left ventricle was cut into small pieces and snap-frozen in liquid N2. Four samples per genotype were pooled to obtain approximately 80 mg of heart tissue per sample, into gentleMACS M tubes containing Lysis Buffer (5 mM CaCl2, 3 mM MgAc, 2 mM EDTA, 0.5 mM EGTA, and 10 mM Tris-HCl pH 8 in H2O, supplemented prior to use with 1 mM DTT, 1 µg/mL Actinomycin D, 0.05% Protease Inhibitor Cocktail, and 0.04 U/µL RNA inhibitors-RNAse OUT). Isolation protocol is adapted from Amoni et.al., DOI: 10.1161/CIRCEP.122.011677. The first step of the isolation protocol was a mechanical dissociation using the gentleMACS dissociator. After mechanical dissociation, tissue was exposed to lysis buffer supplemented with NP-40 (0.1%) and digitonin (0.01%) for lysis of the cell membrane. After a 15-minute incubation on ice, the homogenate was passed through a 30 µm filter and spun down. The pellet was dissolved in sucrose buffer (1 M sucrose, 3 mM MgAc, 10 mM Tris-HCl pH 8 in H2O, supplemented with 1 mM DTT, 1µg/mL Actinomycin D, 0.05% Protease Inhibitor Cocktail and 0.04 U/µL RNA inhibitors-RNase OUT). To isolate the nuclei from the cell debris, the nuclei-containing sucrose solution was pipetted gently on top of fresh sucrose buffer. After centrifugation, nuclei were pelleted followed by multiple rounds of resuspension in washing buffer (750 µg/mL UltraPureTM BSA, 0.04 U/µL RNA inhibitors-RNAse OUT in PBS) prior to sorting. Nuclei were sorted on an ARIA3 FACS sorter based on 7-AAD staining. Sorted nuclei were counted on a LUNA cell counter, spun down and resuspended in washing buffer to the desired concentration according to the 10X Genomics recommendations. According to 10X Genomics. After FACsorting, samples were submitted to the KU Leuven Genomics Core and processed using the 10X Genomics Next GEM Single cell multiome Assay for Transposase-Accessible Chromatin (ATAC) + Gene Expression Reagents kit to generate the Gel beads in Emulsion (GEMs) containing single nuclei. Next, joint single nuclei RNA and single nuclei ATAC libraries were generated according to the 10X Genomics protocol. Finally, libraries were sequenced at a depth of 30K on an Illumina Novaseq 6000 by the KU Leuven Genomics Core.