Single cell sequencing of fibroblast-like synoviocytes (FLS) isolated from hind paws of Col6a1-cre Bmal1-flox PER2::LUC mice
Single cell sequencing of fibroblast-like synoviocytes (FLS) isolated from hind paws of Col6a1-cre Bmal1-flox PER2::LUC mice
Hind paws were dissected to remove skin and separate joints, then incubated with collagenase IV (10 mg/ml, Sigma C5138) in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37ºC with shaking for 1-1.5 hours.  The cell suspension was passed through a 40 micron filter then cells were centrifuged and the pellet washed with PBS.  Podoplanin positive cells were isolated using the EasySep Release Mouse PE Positive Selection kit (Stemcell, 17656) and a Podoplanin-PE conjugated antibody (BioLegend 127407), according to the manufacturer's instructions.  Eluted cells were immediately fixed using the Parse Cell Fixation kit (Parse Biosciences SB1003).  Fixed cell suspensions were counted using a haemocytometer before being flash frozen in liquid nitrogen and stored at -80ºC prior to barcoding and library preparation.     Fixed cells were barcoded using the Evercode Whole Transcriptome Mini kit (Parse Biosciences EC-W01010), in accordance with the manufacturer's instructions. Libraries for sequencing were prepared using the Evercode Whole Transcriptome Mini kit (Parse Biosciences EC-W01010), in accordance with the manufacturer's instructions.  Library fragment size and quality was assayed using a 4200 TapeStation (Agilent Technologies).