Nanodrop lite

We initially confirmed the expression of 7 key m6A regulatory factors in GSE74089. Then, we collected 6 femoral head specimens from the Third Affiliated Hospital of Guangzhou University of Chinese Medicine, comprising three with ONFH and three with femoral neck fractures (FNF). Approval for this study was granted by the Ethics Committee of the Third Affiliated Hospital, and patient informed consent was obtained. The samples were obtained during total hip arthroplasty. The bone tissue was immediately frozen in liquid nitrogen, and RNA extraction followed a standardized protocol. Thermo Scientific's qRT-PCR quantified mRNA transcripts using the NanoDrop Lite and CFX96 Touch real-time PCR detection systems (BIO-RAD, CFX96, USA). Amplification conditions comprised an initial 95 °C for 10 min, succeeded by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 60 °C for 30 s. The stability of potential candidate gene expression was evaluated using the standard comparative method (ΔΔCt), with relative target gene expression levels computed using the 2 − ΔΔCt method. Primer sequences are shown in Additional file 1: Supplementary Table 1.

After 2, 7, or 14 days of culture, chips were placed in an Eppendorf tube containing 400 µL TRK lysis buffer (Omega Bio-tek, Norcross, GA, USA, HCR003) with 8 µL β-mercaptoethanol (EMD Millipore, Burlington, MA, USA, 444203) and stored at −80 °C. For RNA extraction, chips were thawed and vortexed vigorously to dislodge the cell contents out of the MBs. The solution was transferred to homogenizer tubes (Omega Bio-tek, Norcross, GA, USA, HCR003) and extracted using the E.Z.N.A Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA, R6834). RNA was quantified with a spectrophotometer (NanoDrop Lite) and transcribed to cDNA with the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA, 170-8890) according to the manufacturer’s instructions. qPCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, A25742) with a CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA) using 3 technical replicates per sample. Results were normalized to housekeeping gene Rps29 and day 0 freshly isolated cells and analyzed using the 2^−ΔΔCt method. Primer sequences are shown in Supplementary Table S1.