Endosome-associated lc3b mRNA is translated in axons and the protein contributes to endosomal clearance
Endosome-associated lc3b mRNA is translated in axons and the protein contributes to endosomal clearance
Human neuroblastoma SH-SY5Y cells (Sigma, 94030304) SH-SY5Y cells were cultured in RPMI-1640 Medium (Sigma, R8758) supplemented with 1% MEM non-essential amino acids (EuroClone, ECB3054D), 1% penicillin/streptomycin (EuroClone, ECB3001D), and 10% heat-inactivated fetal bovine serum (EuroClone, ECS0180LH). Cells were split twice a week, using 1X trypsin-EDTA (EuroClone, ECB3052D) and used up to passage 35. All cells were maintained in a 5% CO2, 37 °C humidified environment. SH-SY5Y cells were incubated in 500 μM biotin phenol (Chemodex, B0270) in their original medium for 30 min. To favor a proper diffusion, biotin phenol was first diluted in half of the medium and then added to the cells. 1 mM H₂O₂ (Sigma, H1009) was added in the same way to the cells and left in incubation for 2 min with gentle agitation. To block the APEX2 reaction, all medium was then removed, and cells were washed with quencher solution [10 mM Sodium Azide (Sigma, S2002), 10 mM Sodium Ascorbate (Sigma, A4034), 5 mM Trolox (Sigma, 238813) in PBS]. For immunoprecipitation of biotinylated RNAs, cells were quickly washed once with ice-cold quencher solution and then three times for 5 min with ice-cold quencher solution without Sodium Azide. Following biotinylation, cells were scraped in 600 μL of buffer RLT Plus supplemented with 10 μl/ml B-mercaptoethanol (Sigma, M6250). Once collected, samples were mechanically lysed by passing them three times through a 25-gauge needle, fitted to an RNase-free syringe. Homogenized samples were then processed following the RNeasy Plus Mini Kit manufacturer's instructions (QIAGEN, 74134).  RNA libraries were prepared using the Illumina Ultra-Low Input RNA Library Preparation Kit, following the manufacturer's recommended protocol. This kit is specifically designed for transcriptome analysis from samples containing very limited amounts of RNA, enabling reliable cDNA synthesis and library generation from ultra-low input material.