Multi-omics and 3D-imaging reveal bone heterogeneity and unique calvaria cells in in mice and humans

Version 1

Sample preparation for proteomics analysis was performed as described previously with slight modifications. Briefly, for mouse samples, SDC lysis buffer (2% SDC, 100 mM Tris-HCl pH 8.5) was used to lyse the cell pellets at 95°C for 45 min at 600 rpm in a thermoshaker. For human samples which were fixed in PFA, prior to the SDC lysis buffer step, the samples were first resuspended in 6% SDS buffer, heat denatured, sonicated and then precipitated using 80% acetone overnight in -20°C. Next day, these samples were centrifuged and the pellet was resuspended in SDC lysis buffer. After this, the procedure remains the same for both mouse and human samples. The samples in SDC buffer were sonicated in high mode for 15 cycles (30 sec OFF, 30 sec ON) (Bioruptor® Plus; Diagenode). The samples were again heated at 95°C for 45 min at 600 rpm in a thermoshaker. The extracted and solubilized protein concentration was estimated by BCA method and 25 µg of protein was further reduced and alkylated using a final concentrations of 10 mM TCEP and 40 mM CAA in dark, at 45°C for 10 min with 600 rpm in a thermoshaker. The protein samples were digested overnight with Trypsin and LysC (1:50, protease:protein ratio) at 37°C, 1,000 rpm shake. Resulting peptides were acidified with 1% TFA 99% isopropanol with 1:1 volume-to-volume ratio, vortexed and centrifuged to pellet residual particles. The supernatant was transferred to fresh tubes and subjected to in-house built StageTip clean-up consisted of three layers of styrene divinylbenzene reversed-phase sulfonate (SDB-RPS; 3 M Empore) membranes. Peptides were loaded on the activated (100% ACN, 1% TFA in 30% Methanol, 0.2% TFA, respectively) StageTips, run through the SDB-RPS membranes, and washed by EtOAc including 1% TFA, isopropanol including 1% TFA, and 0.2% TFA, respectively. Peptides were then eluted from the membranes via 60 µL elution buffer (80% ACN, 1.25% NH4OH) and dried using vacuum centrifuge (40 min at 45°C). Finally, peptides were reconstituted in 10 µL of loading buffer (2% ACN, 0.1% TFA) and peptide concentration was estimated using PierceTM Quantitative Colorimetric Peptide Assay.