High speed low temperature tissue homogenizer

Total RNA was extracted with Trizol (Invitrogen, CA, USA) from mouse aortas (n = 10 per group) using a high‐speed low temperature tissue homogenizer (Servicebio) at 60 Hz for 3 min at 4 °C. The RNA integrity number was inspected for RNA integrity using an Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, US). Total RNA was further purified and qualified with an RNAClean XP Kit (Beckman Coulter, CA, USA) and RNase‐Free DNase Set (QIAGEN, GmBH, Germany).
Strand‐specific libraries were prepared with a VAHTS Universal V6 RNA‐seq Library Preparation Kit for Illumina (Vazyme, Nanjing, China) so that strand‐specific libraries could be prepared according to the manufacturer's instructions. Sequencing read counts were calculated with Stringtie (v.1.3.0).^[99] Then the expression level was normalized from different samples using the trimmed mean of M values method,^[100] and converted the normalized expression levels of samples into fragments per kilobase of transcript per million mapped fragments. The difference of gene expression between groups was analyzed, the P‐values were calculated, and multiple hypothesis tests were performed with the edgeR^[101] (v.3.32.1) package of R. GO enrichment analysis (http://www.geneontology.org/) was performed with false discovery rate ≤0.05 as a threshold.

The femurs were collected from wild-type, Mir155-Tg, and Mir155-KO mice. And bones were ground with a high-speed low-temperature tissue homogenizer (Servicebio, China). As previously reported (Teng et al., 2022 (link)), the miRNAs were extracted from BMSCs and ground bone tissues with the MolPure Cell/Tissue miRNA Kit (Yeasen, China) as per the manufacturer’s instructions. The miRNA was further reversed by Tailing reaction using miRNA first strand cDNA synthesis kit (Accurate Biology, China). In brief, 3.75 μL miRNA, 5 μL 2×miRNA RT Reaction Solution, 1.25 μL miRNA RT Enzyme Mix were incubated at 37°C for 1 hr, 85°C for 5 min. The mRNAs from osteoclasts induced from Mir155-KO and wild-type mice were extracted with an RNA extraction kit (Accurate Biology, China) as per the manufacturer’s instruction. Total RNA (500 ng) was transcribed with reversed regents (Accurate Biology, China). RT-qPCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China) on an AriaMx Real-time quantitative PCR machine (Agilent, USA). The PCR conditions were 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. The fold change relative to the control group was measured by the 2^-∆∆Ct method. The primers used were shown in Table 1.