GSM5526268: Wild type FAPs 2; Mus musculus; RNA-Seq
FAPs were isolated from the hindlimb muscles of 8Mo B6A/J and WT female mice in a modified protocol from our previously published study (Hogarth et al, NComms 2019). Mice were euthanized and the tibialis anterior, extensor digitorum longus, gastrocnemius, soleus, quadriceps and psoas were immediately dissected. Non-muscle tissue including tendon, nerve and overlying fascia were carefully removed, and muscles were minced finely in a sterile dish and incubated in Collagenase II (2.5U/ml, ThermoFisher, #17101015) in PBS for 30min at 37°C. The resulting slurry was washed with sterile PBS before further digest in Collagenase D (1.5U/ml, Sigma Aldridge, #COLLD-RO) and Dispase II (2.4U/ml, Sigma Aldridge, #D4693) in PBS for 60min at 37°C. Resulting slurries were passed through 100µm and 40µm strainers and primary cells were resuspended in 1ml PBS with 2%FBS and 2nM EDTA. The suspensions were subsequently stained with anti-PDGFRα-APC (1.0 µg per 106 cells in 100 µl, Biolegend, #135908) for flow cytometry. Immediately prior to FACS, cells were stained with 7AAD viability staining solution (1:200 dilution, BioLegend #420404). Cell suspensions were then sorted on an Influx cell sorter (Becton Dickinson, #646500); live cells were enriched by gating based on forward and side scatter, and doublets were excluded using trigger pulse width gating. We gated out 7AAD+ cells to further increase sample viability, before purified PDGFRα+ FAPs were then collected for single cell sequencing. Libraries were prepared according to 10x's instructions accompanying the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (PN-1000075 ). At the 10x controller step, single-cell suspension at a density of 1000 cells/µL in 1x PBS with 0.04% BSA was added to real-time polymerase chain reaction (RT-PCR) master mix aiming for sampling around 8000 cells, and then loaded together with Single Cell 3′ v3 gel beads and partitioning oil into a Single Cell 3′ Chip according to the manufacturer's instructions. RNA transcripts from single cell were uniquely barcoded and reverse-transcribed within droplets to generate single cell cDNAs. Single cell cDNA libraries were generated via end-repair, A tailing, ligation (Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, PN-1000075) and index PCR ( Chromium i7 Multiplex Kit, PN-120262) according to the manufacturer's instructions (10x genomics, US). Sizes of the preamplified cDNA and sequencing libraries were determined by the DNA high sensitivity kit (Agilent, Cat #5067-4626). All single-cell libraries were sequenced with a customized paired-end with single indexing (28/8/91-bp for v3 libraries) format according to the recommendation by 10X Genomics. These libraries were sequenced on a Novaseq 6000 system using the S1 flow cell (Illumina, US). Droplet based single cell RNA seq