biostudies-arrayexpressStefano de PretisMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15783The primary objective of this project was to identify transcripts in close proximity to endosomes using the APEX-seq approach. To achieve this, we employed SH-SY5Y cells expressing APEX2-NES, APEX2-RAB5A, or 2xFYVE-APEX2 constructs. Biotinylation of RNAs was validated by dot blot, and biotinylated RNAs were enriched via streptavidin immunoprecipitation and subsequently analyzed by RNA sequencing. This approach enabled us to identify mRNA encoding the LC3B protein in association with endosomes.biostudies-arrayexpressSample Treatment - 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.Library Construction - 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.Growth Protocol - 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.Sequencing - Sequenced on Illumina HiSeq 2500, 150 bp paired-end reads.Sample Collection - Human neuroblastoma SH-SY5Y cells (Sigma, 94030304)Nucleic Acid Extraction - 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).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - FASTQ sequencing reads were adaptor-trimmed and quality-filtered with Trimmomatic (Bolger et al., 2014). Filtered reads were mapped to the mm10 mouse reference genome (https://www.gencodegenes.org/mouse/) with STAR (Dobin & Gingeras, 2016).Data Transformation - Gene counts were obtained using featureCounts (Liao et al., 2014). Raw reads were normalized and tested using the DESeq2 R package.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2500RNA-seq of coding RNAMus musculusLocal translation of endosome-associated lc3b mRNA in axons contributes to endosomal clearance.Pappacena F, Caterino C, De Pretis S, Sironi C, Esposito A, Cesare G, Bruni M, Cioni JM.Francesca PappacenaCristina SironiJean-Michel CioniMichela BruniCinzia CaterinoStefano de PretisfalseEndosome-associated lc3b mRNA is translated in axons and the protein contributes to endosomal clearanceThe primary objective of this project was to identify transcripts in close proximity to endosomes using the APEX-seq approach. To achieve this, we employed SH-SY5Y cells expressing APEX2-NES, APEX2-RAB5A, or 2xFYVE-APEX2 constructs. Biotinylation of RNAs was validated by dot blot, and biotinylated RNAs were enriched via streptavidin immunoprecipitation and subsequently analyzed by RNA sequencing. This approach enabled us to identify mRNA encoding the LC3B protein in association with endosomes.2026-09-01T00:00:00Z2026-09-01T08:20:10.128Z2025-10-20T11:10:07.379ZE-MTAB-15783publ-0-r68h-removableERP182496EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1038/s44319-026-00867-5