title: GSM9463149: steroid-induced osteonecrosis of the femoral head, sample2; Homo sapiens; RNA-Seq
library_construction_protocol: Each femoral head was washed once with PBS and cut into quarters to facilitate mechanical digestion. Approximately 20-25g (wet weight) of processed tissue was divided into 4-5 tubes (5g per tube) for enzymatic digestion. Tissue fragments were digested in a solution containing 0.2% collagenase type II (Sigma-Aldrich) and 0.2% dispase II (Sigma-Aldrich) in serum-free Dulbecco's Modified Eagle Medium (DMEM) at 37°C for 4 hours with continuous agitation. The digested suspension was then filtered through a 70-μm strainer, and the resulting filtrate was centrifuged at 400 × g for 5 minutes. The cell pellet was resuspended in 20 ml of serum-free DMEM. To remove bone debris and enrich for nucleated cells, the cell suspension was subjected to density gradient centrifugation using a Percoll solution (Sigma-Aldrich). RBC lysis was performed using a commercial buffer (Lonza, Basel, CH). Cells were then stained with anti-RBC antibody according to manufacturer's protocol (BioLegend, San Diego, CA, USA), washed, and resuspended in PBS with 1% FBS containing DAPI. Fluorescence-activated cell sorting (FACS) analysis was performed to identify and sort live cells using a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Single-cell samples were prepared using the Chromium Next GEM Single Cell 3/ Reagent Kits v3 and Chip Kits (10x Genomics, Pleasanton, CA, USA), following the manufacturer's protocol. In brief, FACS-sorted live cells were resuspended in 0.04% BSA-PBS solution. Approximately 700–1000 cells/μL were added to each chip well, aiming for a target cell recovery of 5000–9000 cells. Gel bead-in emulsions (GEMs) were generated using the Chromium controller (10x Genomics). GEMs underwent reverse transcription, the droplets were broken, and single-stranded cDNA was isolated. The cDNA was cleaned using DynaBeads and amplified. After amplification, adapters were ligated to the cDNA, followed by further amplification and cleanup with SPRIselect. The quality of the purified libraries was checked using Agilent Tapestation D1000 (Agilent, Santa Clara, CA, USA) and their concentration was quantified by dsDNA high-sensitivity kit using Qubit fluorometer (Thermo Fisher Scientific, MA, USA). Equimolar concentration of the libraries was pooled and sequenced on Illumina NextSeq platform (Illumina, San Diego, CA, USA).