title: GSM8189239: FD4, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh lesion specimens were finely minced into small particles until they reached the consistency of sand and digested with 2mg/ml collagenase D solution (Roche, Mannheim, Germany) in basal medium Dulbecco's Modified Eagle medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 1% antibiotic-antimycotic solution, containing 10,000U/mL penicillin and 10,000U/mL streptomycin) (Life Technologies) for 4h at 37°C with gentle agitation. Digestion was terminated with fetal bovine serum (FBS; Life Technologies) and washed three times and resuspended in freshly prepared growth medium (DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution). The resulting cell suspension was passed through a 70-mm cell strainer, pelleted by centrifugation at 300 x g, resuspended with 5ml of 1x RBC Lysis Buffer (Invitrogen, San Diego, CA), washed with DMEM, and pelleted for cryopreservation in cryopreservative medium (Nippon Zenyaku Kogyo, Fukushima, Japan) or culture in a 75cm2 T-flask containing growth medium at a density of 3x105 cells per flask in an atmosphere of 37°C, 5% CO2, and 95% humidity. Any undigested residues were transferred to a 75cm2 T-flask and maintained in growth medium for primary explant culture under normal culturing conditions. Approximately 2 weeks after explantation when outgrown cells reached near confluence, cells and tissue fragments were detached using 0.25% trypsin-EDTA (Gibco, Paisly, Scotland), washed, and resuspended in culture medium. Cells were collected from the suspension using a 70-mm cell strainer (PluriSelect, San Diego, CA) and either cryopreserved or replated in fresh culture dishes. Normal bone specimens consisted of marrow which was used to isolate bone marrow mesenchymal stromal cells (BMSCs). The marrow was scraped into the basal medium prepared as described above, followed by pipetting and serial passages through needles of decreasing diameter. Cells were treated with RBC Lysis Buffer prior to being either cryopreserved or plated into 75cm2 T-flask at a density of 3x105 cells per flask containing growth medium (DMEM supplemented with 10% FBS and 1% antibiotic-antimyotic) and cultured until near confluence. Cryopreserved cells dissociated from specimen digestion and cells obtained after two weeks of explant culture (2x106 cells each) were used for scRNAseq analysis. The cell concentration of the single-cell suspension was maintained at500cells/ul(Total 1 * 10^5 cells)and loadedontoChromium Next GEM Chip G Single Cell Kit(10X genomics, cat no.1000120). Single-cell RNA-seq (scRNA-seq) libraries were constructed usingChromium Next GEM Single Cell 3' Kit v3.1(10X genomics, cat no.1000268).Individual libraries were diluted to250-500 pMand sequencing was completed on the IlluminaNextSeq 500sequencing platform,using S2ReagenttKit(200cyc)paired-end mode.