Translation efficiency analysis in MIN6 cells using AHARIBO kit

The AHARIBOTM kit (IMMAGINA BioTECHNOLOGY) was used to isolate mRNA associated to active ribosomes. Briefly, MIN6 cells were seeded in 6 well plate, transfected with ASOs of Ctr or targeting 5'tRF-Glu(CTC) (a-GLU) and treated with BSA or palmitate for 48h. On the day of collection, the cells were incubated for 40 min with methionine-free complete DMEM and then supplemented withl-azidohomoalanine (AHA) methionine analog for 10 min followed by 5 min sBlock treatment to immobilize nascent peptides on the active ribosomes. Upon cell lysis, AHA-labelled nascent peptides were used as tags to bind magnetic beads with a click chemistry reaction and to purify ribosomes complexed to mRNA in translation. RNA extracted from total cell lysates was used as input. RNA sequencing was then performed with mRNA pulled-down (PD, in translation) and with inputs. . Library preparation was performed with the Illumina Stranded mRNA Library Prep kit and sequenced with an AVITI instrument using the SBS chemistry v4 (Illumina). After demultiplexing, reads annotation and normalization were performed using the QIAGEN RNA-seq Analysis Portal v 5.1. Translatome analysis was performed using the limma R package (REF). Translation efficiency (TE) was calculated by performing the ratio between PD and input mRNA counts. The dataset contains normalized transcript counts, ratio and fold change calculations for the comparisons PA vs BSA in Ctr transfected cells or PA vs BSA in a-GLU transfected cells.