Thermo nanodrop lite

The concentration of the pUC57-EGFP, pMD18-B646L, and pUC57-mCherry standard plasmids was determined using the Thermo Nanodrop Lite apparatus (Thermofisher Scientific, Shanghai, China), and then serial dilutions (10 folds) were performed. The diluted plasmids were then subjected to triplex RT-PCR. The copy numbers of pUC57-EGFP, pMD18-B646L, and pUC57-mCherry were determined according to the DNA copy number calculation formula [dsDNA copy number (copies/mL) = 6.02 × 10²³(copies/mol) × concentration (g/mL) / DNA length × 660], which were 1.17 × 10¹¹ copies/μL, 1.60 × 10¹¹ copies/μL, and 1.00 × 10¹¹ copies/μL, respectively.

PAMs, PK15, and 3D4-21 cells were harvested from T75. The total RNA was extracted from the cells using RNAiso Plus (TAKARA, Kyoto, Japan), according to the manufacturer’s instructions. RNA quantity and purity were assessed using a Thermo NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, MA, USA). Samples were sent to Novogene (Beijing, China) for the removal of ribosomal RNA to construct strand-specific libraries. First, ribosomal RNA was removed from total RNA, and RNA was fragmented into 250–300 bp sections. The fragmented RNA was used as a template and a random oligonucleotide was used as a primer to synthesize the first strand of cDNA. Thereafter, RNase H was used to synthesize the first strand of cDNA. The RNA strand was degraded, and the second strand of cDNA was synthesized using dNTPs (dUTP, dATP, dGTP and dCTP) as raw materials under the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and connected to sequencing adapters, and AMPure XP beads were used to screen cDNAs of approximately 200 bp. The U-containing cDNA second strand was then degraded using USER enzyme, and finally PCR amplification was performed to obtain a library.

RAW264 cells were prestimulated with 50 ng/mL RANKL for 24 h. Then, the cells were stimulated with 1 μg/mL Mfa1 and FimA fimbriae or 50 ng/mL RANKL for 48 h. Total RNA was then extracted using NucleoSpin RNA (Macherey-Nagel Inc, Bethlehem, PA, USA) in accordance with the manufacturer’s protocol. The sample concentration was measured by a Thermo NANO DROP LITE (Thermo Fisher Scientific) and underwent cDNA conversion using a Biosystems GeneAmp PCR System (Thermo Fisher Scientific). Conditions were 37 °C for 15 min, 50 °C for 5 min, 98 °C for 5 min, and hold at 4 °C. Then, to quantify mRNA expression, real-time qPCR was performed using Taqman gene expression assays (Thermo Fisher Scientific) for mouse Acp5 (Trap) (Mm00437135-m1), Mmp9 (Mm00442991-m1), Ctsk (Mm00484039-m1), and Nfatc1 (Mm00479445-m1) with TaqMan Universal PCR Master Mix (Thermo Fisher Scientific). mRNA levels were normalized to eukaryotic 18S rRNA (Hs99999901_s1). qPCR was performed using a StepOnePlus™ Real-Time System (Thermo Fisher Scientific). The thermocycling conditions were 40 cycles of 10 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. Relative changes in gene expression were calculated using the 2^−ΔΔCt method. 18S rRNA (Hs99999901-s1) was used as an internal control.