Qubit fluorometric assay

To perform single-cell RNA sequencing (scRNA-seq), cells isolated through fluorescent activated cell sorting (FACS) were loaded onto a Chromium Single Cell Chip (10x Genomics, United States) according to the manufacturer’s protocol. The perdurance of the GFP in the Tg(gfap:GFP)^m2001 enables us to track all glia derived cells including those that have de-differentiated into progenitors as well as those that start differentiating into mature cells (glia and neurons). The scRNA-seq libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3’ Library and Gel Bead kit v2 and v3 Chip kit (10x Genomics, 120,237). Library quantification and quality assessments were performed by Qubit fluorometric assay (Invitrogen) and dsDNA High Sensitivity Assay Kit (AATI, DNF-474-0500). Analysis of DNA fragments was performed using the High Sensitivity Large Fragment -50 kb Analysis Kit (AATI, DNF-464). The indexed library was tested for quality and sequenced using an Illumina NovaSeq 6,000 sequencer with the S2 flow cell using paired-end 150 base pair reads. Sequencing depth was 60 K reads per cell.

A total of nine littermate mice, including miR-17~92 cKO, miR-17~92^fl/fl and agomir-17-treated miR-17~92 cKO mice, were used in this experiment. Mice in the agomir-17 group received weekly injections of agomir-17 (2 nmol/injection) in the knee joints for a total of 4 injections. After mice were euthanized, the medial femoral condyle, including the articular cartilage, subchondral bone and adjacent bone marrow cavity, was dissected using scissors to ensure that intact cartilage tissue was obtained. The tissue was cut into small pieces and was then digested at 37 °C for 2 h in 10 mL of DMEM containing 0.2% NB4 collagenase and 0.3 mg/mL DNase I (Thermo Scientific). The mixture was filtered through a 40-μm strainer and centrifuged at 300 × g for 10 min. Cells were counted using a hemocytometer, and ~8000 cells in 25 μL of PBS per group were used for experiments. Single cells were processed on a Chromium Single-Cell Platform with a Chromium Single-Cell 3′ Library and Gel Bead Kit v2 (10X Genomics, PN-120237) and a Chromium Single-Cell A Chip Kit (10X Genomics, PN-120236) according to the manufacturer’s protocol. DNA quality was assessed with an Agilent Bioanalyzer to ensure transcript integrity and purity, and amount was assessed with a Qubit fluorometric assay (Invitrogen).

The multi-epitope antigens used in the indirect ELISA were obtained according to Santos et al. [18 (link)]. Briefly, the synthetic genes were subcloned into the pET28a vector and expressed in Escherichia coli BL21-Star (ThermoFisher Scientific). Expression was induced with 0.5 μM of IPTG (isopropyl β-D-1-thiogalactopyranoside) and the soluble proteins purified by both ion exchange and affinity chromatography. Finally, the purified muti-epitope antigens were quantified by Qubit fluorometric assay (ThermoFisher Scientific). Fig 1 illustrates the SDS-PAGE of the antigens after purification. Details about IBMP composition has been seen in previous studies of our group [18 (link),19 (link),23 (link)].

Small RNA libraries were created using the New England Biolabs small RNA library protocol (New England Biolabs). Library construction used a two-step ligation process to create templates compatible with Illumina based next generation sequence (NGS) analysis. Where appropriate, RNA samples were quantified using a Qubit fluorometric assay (Thermo Fisher Scientific). RNA quality was assessed using a pico-RNA chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). Library creation uses a sequential addition of first a 3’ adapter sequence followed by a 5’ adapter sequence. A cDNA copy was then synthesized using ProtoScript reverse transcriptase (New England Biolabs) and a primer complimentary to a segment of the 3’ adapter. Amplification of the template population was performed in 15 cycles (94°C for 30 sec; 62°C for 30 sec; 70°C for 30 sec) and the amplified templates were PAGE (polyacrylamide gel electrophoresis) purified (147 bp DNA) prior to sequencing. All NGS libraries were indexed. The final concentration of all NGS libraries was determined using a Qubit fluorometric assay and the DNA fragment size of each library was assessed using a DNA 1000 high sensitivity chip and an Agilent 2100 Bioanalyzer. Sequence analysis was performed using the rapid run platform and single end 50 base sequencing by synthesis on an Illumina Hi-Seq 1500 using the TruSeq SBS kit v3.