GSM9135778: mixed 'Patient_bone_femoral_head_biopsy' and 'scaffold_populated_with_patient_PBMC or _BMMNC'; Homo sapiens; RNA-Seq
Single-nuclei RNA sequencing was performed in single replicates of two primary BOAC systems colonized with BMMNCs (BM-BOAC), or with PBMCs (PB-BOAC), as well as a biopsy from the same donor serving as a reference. Single nuclei were extracted from fixed (using 10X Genomics tissue fixation protocol), frozen sample by a combination of physical disruption into small pieces and incubation at 37°C for 10 min in dissociation buffer (1 mg/mL Liberase TH, Merck, Darmstadt, Germany) in RPMI 1640 (Gibco/Thermo Fisher Scientific, MA, USA) before filtering through a 10 µm pluriStrainer (pluriSelect Life Science, Leipzig, Germany). Single-nuclei suspensions were subsequently pelleted by centrifugation (850 x g, 5 min, RT) the supernatant was discarded and nuclei pellets were resuspended in quenching buffer (10X Genomics, Pleasanton, CA, USA). library construction was performed according to manufacturer's instructions (10X Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit). The samples were incubated with differently barcoded Chromium Human Transcriptome Probe Sets (10X Genomics, Pleasanton, CA, USA) for multiplexing and a single-nuclei RNA seq library was generated using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit on a Chromium X device (10X Genomics, Pleasanton, CA, USA). Libraries were quantified and library fragment size was determined using a Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, MA, USA) and a HS DNA Kit assay on a 2100 Bioanalyzer device (Agilent).