Single cell RNA-seq of human enthesis from healthy and inflamed tissues
Single cell RNA-seq of human enthesis from healthy and inflamed tissues
Biopsies were taken either at autopsy or during surgery. The instrument was inserted to bone contact and then gently withdrawn to obtain the biopsy (5 mm) 5 mm above the bone. Biopsies were immediately transferred into saline. Entheseal tissues were then minced and digested in RPMI 1640 containing 2 mg ∙ ml−1 collagenase D (Roche), 0.2 mg ∙ ml−1 Dispase II (Sigma-Aldrich) and 0.2 mg ∙ ml−1 DNase I (Roche). Samples were incubated three times at 37 °C for 20 min with constant shaking (2,000 rpm) on a thermal shaker (Eppendorf). After 20 min, the supernatant was collected, filtered (70 µm) and fresh digestion medium was added to the samples. The digestion was repeated for a total of 3 times. RBC lysis was performed using RBC lysis buffer (BioLegend) according to the manufacturer's recommendations. Cells were washed, counted and concentrated to 1,000 cells / µl.    15,000 and 1,000 cells from healthy or inflamed enthesis were loaded into a single well of a Chromium chip G, respectively (10x Genomics) immediately after tissue dissociation and further processed according to standard Chromium Next GEM Single Cell 3' Gene Expression protocol (10x Genomics protocol CG000316 Rev C).   3' gene expression library was generated using Chromium Next GEM Single Cell 3' Kit 3.1 Library Construction Kit . 