Age-Associated Loss of Lymphatic Vessels Promotes Cardiac Inflammation (sc-RNA-SEQ)
Age-Associated Loss of Lymphatic Vessels Promotes Cardiac Inflammation (sc-RNA-SEQ)
Aged male and female C57Bl/6J wildtype mice were purchased from Janvier (Le Genest SaintIsle, France) and from Charles River (Sulzfeld, Germany). Homozygosity of these inbred mice was controlled by Janvier and Charles River using exome sequencing. Mice have been kept in a 12h-day-night cycyle. All animal experiments have been executed in accordance to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the local authorities by the state of Hesse (Regierungspräsidium Darmstadt). Resulting non-cardiomyocyte cellular suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol. Murine scRNA-seq libraries were prepared using Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics), according to manufacturer's protocols. Briefly, the initial step consisted in generating cell partitioning droplets where individual cells were isolated together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (37002D, Thermo Fisher Scientific). Bulk cDNA was amplified using a Biometra Thermocycler Professional Basic Gradient with 96-Well Sample Block (98 °C for 3 minutes; cycled 14×: 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 minute; 72 °C for 1 minute; held at 4 °C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (B23318, Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 Reagent Kit as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads. Single cell RNA libraries were generated using the Gem-X Kit (1000686, 10X Genomics), using standard GEM construction, amplification, fragmentation and indexing procedures.