Illumina HiSeq 4000 sequencing; Transcriptional disorganization of fibroblasts is a hallmark of the aging heart
Transcriptional disorganization of fibroblasts is a hallmark of the aging heart
Mice were obtained from Janvier (Le Genest Saint-Isle, France) and all animal experiments were approved by the Regional Board of the State of Hessen, Germany. The obtained hearts were either snap frozen with liquid nitrogen for nuclei isolation or freshly used for cell isolation.  Cross-sections of mouse hearts (derived from three 12 weeks old and from three 18 months old mice) were dissociated in liquid nitrogen using a mortar. The sample was then transferred into a Dounce homogenizer. For isolation of nuclei we strictly followed the experimental steps that are detailed in 3. Samples were homogenized and lysed to release the nuclei, followed by single-cell FACS (Aria III, BD Genomics) sorting of DAPI stained high quality nuclei to sort out any residual cell debris. Integrity and purity of the nuclei was inspected visually using a Leica DMi8 inverted microscope. Yield of nuclei was quantified by using a Neubauer chamber.  Generation of libraries for single-cell mRNA sequencing was done using the Chromium Single Cell 3' protocol (10X Genomics). Briefly, we applied droplet based nuclei encapsulation, lysis, as well as individual transcriptome (UMI) and nuclei barcoding for generation of 3' end-counting mRNA sequencing libraries, using the Single Cell 3' v2 Reagent Kits from 10X Genomics.
Experimental Factor: age
12