GSM9409334: TVWB29; Mus musculus; RNA-Seq
Tibialis Anterior (TA), gastrocnemius and quadriceps muscles of 7 months post-AAV injection of two individuals per genotype (NaCl-, GFP-, ATG polyG-GFP-, uGIPpolyG-GFP-, uN2CpolyG-GFP- or LOC6polyG-GFP- injected mice) were isolated and dissociated with scissor in hypotonic buffer (250 mM sucrose, 10 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH8, 25 mM HEPES, 0,2 mM PMSF, 0,1 mM DTT, 0,3% Triton X-100, 0,2 U/µL RNase inhibitor). The muscles were homogenized with Precellys 24 Touch (Bertin) for 25 sec at 5000 rpm. Samples were filtered through 100 µm and 20 µm filters and centrifuged 10 min at 400 x g at 4°C. Pellets were washed in PBS, 2% BSA, 0,2 U/µL RNase inhibitor, centrifuged 10 min at 400 x g at 4°C, resuspended in PBS, 2% BSA, 0,2 U/µL RNase inhibitor, 200 nM DAPI to sort nuclei with FACSAria Fusion or FACSAria II. More than 3 million of isolated nuclei were centrifuged 12 min at 400 x g at 4°C and fixed with formaldehyde using Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN 1000414) following 10X genomics procedure (CG000478). P3250 : Single nuclei libraries were generated using the Chromium Next GEM Single Cell Fixed RNA reagent set (10x Genomics, Leiden, The Netherlands), according to the manufacturer's recommendations. During this process, cell (or nucleus) counting was performed using an ADAM MC Plus Fluorescence cell counter (NanoEntek, Seoul, Korea) with a mixed solution of Acridine Orange and 4',6-diamidino-2-phénylindole (DAPI) or by a Trypan Blue exclusion assay on a Buerker Chamber. For each of the 8 samples, from 133000 to 1060000 starting nuclei were hybridized with an unique Mouse WTA Probe Barcode (10x Genomics, PN-1000492). Hybridizations were completed in 80 µL of hybridization mixture plus 20 µL of WTA probes at 42oC for 21h. Following cell re-counting, an equal number of nuclei from each hybridization reaction was combined in a single pool before washing. After re-counting, nuclei were then loaded onto the Chromium iX using a Next GEM Chip Q, targeting 5000 nuclei per hybridization reaction for a total of 96000 nuclei loaded per library (8-plex). Full-length cDNA amplification was completed using 8 cycles of PCR. Dual-index library construction was performed with 10 cycles during sample index PCR. Final library quantification and quality control were performed using the Bioanalyzer 2100 (Agilent Technologies, Les Ulis, France).