Bulk RNA-sequencing of C57BL/6J bone marrow stromal cells stained with anti-Neo1 or anti-TrkB antibodies
Bulk RNA-sequencing of C57BL/6J bone marrow stromal cells stained with anti-Neo1 or anti-TrkB antibodies
For stromal cell collection, tibias, hip bones and flushed femurs were cut into pieces and incubated with digestion buffer (2 mg/ml Collagenase IV and 1mg/ml Dispase II in HBBS, for catalgog numbers please refer to original manuscript) at 37°C for 10 minutes with intermittent shaking. Bone lysate was filtered through a 100 µm strainer and the digestion was quenched with FACS buffer (2% FCS in PBS). This was repeated a total of three times. Cells were incubated in 1ml ACK lysis buffer at room temperature for 10 minutes for red blood cell lysis. Cells were then incubated with FcR blocking agent (anti-CD16/32) at 4°C for 30 minutes to reduce unspecific binding. Cells were next washed twice using MACS buffer (2% FCS and 1mM EDTA in PBS) and incubated with CD45 antibody-coated beads in MACS buffer on ice for 15 minutes for CD45-depletion. Cells were loaded onto MACS LS columns (Miltenyi Biotec, 130-042-401) and flow-through was collected. Lastly, cells were stained with Live-Dead staining. Cells from 12 mice were pooled to a total of four samples (3 mice per sample), then split into two fractions, each. The Neo1 fraction was incubated with conjugated antibodies against CD45, Ter119, CD31, and Neo1 (for antibody catalog numbers, see original manuscript) for 60 minutes at 4°C. Live, single, CD45-/Ter119-/CD31- cells were sorted into positive and negative fractions per sample: Neo1+ and Neo1-. Total RNA was extracted using the PicoPure™ RNA Isolation Kit (Arcturus, KIT0204) protocol for RNA extraction from cell pellets, starting from step 4. DNA was removed using the RNase-Free DNase Set (Qiagen, 79254). 360 pg of total RNA per sample were used for cDNA generation with the SMART-Seq® Total RNA Library Prep with ZapR® Depletion (with UMIs) Kit (Takara, 634354). RNA (RIN >7) was fragmented at 94 °C for 4 min. The Unique Dual Index Kit (1–24) (Takara, 634756) and AMPure XP Beads for DNA Cleanup, 60 mL (Beckman Coulter, A63881) were additionally used. cDNA quality was assessed using Agilent 4200 TapeStation High Sensitivity D5000 reagents (Agilent Technologies, 5067-5593 and 5067-5592) and concentration using the Qubit™ 1X dsDNA High Sensitivity (HS) system (Thermo Fisher Scientific, Q33231 and Q32856). Then, libraries were prepared using NextSeq™1000/2000 P3 XLEAP-SBS Reagents (100Cycles) (Ilumina, 20100990), with 1% NextSeq PhiX Control Kit (Illumina, FC-110-3002) supplied. 