Rabbit anti human igg antibody

sGMSCs were respectively labeled with rabbit anti-human CD14, CD31, CD34, CD45, CD73, CD90, and CD105 (Abcam) according to the manufacturer’s instructions. A rabbit anti-human IgG antibody (Abcam) served as a negative control. The tube was incubated in the dark at room temperature (RT) for 2 h. After incubation, cells were washed three times with PBS to remove unbound antibodies. Subsequently, the cells were resuspended and analyzed using a Beckman CytoFLEX flow cytometer (Beckman Coulter, IN, USA) with appropriate settings and compensation adjustments. The negative control was utilized to establish the negative boundary, while the fluorescence intensity and number of positive cells were measured.

Three µg total RNA was added to 60 µL 5 × IP buffer (50 mM Tris HCl pH 7.4, 750 mM NaCl, 0.5% IGEPAL CA-630) containing 2 µg affinity purified anti-m⁶A rabbit polyclonal antibody (Synaptic Systems, Goettingen, Germany), and then incubated in a rotary shaker for 2 h at 4 °C. A rabbit anti-human IgG antibody (Abcam, Cambridge, UK) was used as negative control. The samples were then mixed with 20 µL sheep anti-rabbit IgG magnetic beads (Invitrogen, Carlsbad, CA, USA) that were blocked in advance with 0.5% BSA for 2 h. The mixture was then incubated at 4 °C for 14 h. The beads were washed three times with 300 µL 1 × IP buffer and twice with 300 µL 1 × wash buffer (100 mM Tris HCl pH 7.4, 50 mM NaCl, 0.1% IGEPAL CA-630). The m⁶A-modified RNA was eluted from the magnetic beads using 300 µL elution buffer (100 mM Tris HCl pH 7.4, 1 mM EDTA, 0.05% SDS, 4 µL proteinase K, 2 µL RNase inhibitor) in the rotary shaker for 1 h at 50 °C. The RNA was extracted by phenol/chloroform/isoamylol (25:24:1) reagent (Tinadz, Beijing, China). The input RNA and m⁶A-modified RNA were both analyzed via RT-qPCR in a 1:1 ratio. The primers are listed in Additional file 2: Table S6. The m⁶A methylation level of lncRNA was calculated using the following formula: $$\mathrm{\Delta }{\text{CT}}_{\text{RIP}}={\text{CT}}_{\text{m6A - IP}}-{\text{CT}}_{\text{Input}}$$ $$\%\text{Input}=2{}^{\wedge }\left(-\mathrm{\Delta }{\text{CT}}_{\text{RIP}}\right)\times 100\%$$