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.本研究采用AHARIBOTM试剂盒（IMMAGINA BioTECHNOLOGY公司）分离与活性核糖体结合的信使RNA（mRNA）。实验简述如下：将MIN6细胞接种于6孔板中，分别转染对照反义寡核苷酸（ASOs）或靶向5'tRF-Glu(CTC)的反义寡核苷酸（a-GLU），随后用牛血清白蛋白（BSA）或棕榈酸处理细胞48小时。样本收集当日，将细胞置于无甲硫氨酸的完全DMEM培养基中孵育40分钟，随后添加l-叠氮高丙氨酸（AHA，甲硫氨酸类似物）孵育10分钟，再经5分钟sBlock处理以将新生肽固定于活性核糖体上。细胞裂解后，以AHA标记的新生肽作为标签，通过点击化学反应（click chemistry）结合磁珠，纯化与正在翻译的mRNA结合的核糖体；以总细胞裂解物中提取的RNA作为输入对照样本。随后对经下拉富集的mRNA（PD，对应正在翻译的组分）及输入对照样本进行RNA测序：文库制备采用Illumina链特异性mRNA文库制备试剂盒，使用AVITI测序仪及SBS化学试剂v4（Illumina公司）完成测序。测序数据经去复接后，通过QIAGEN RNA-seq分析门户v5.1完成读段注释与标准化处理；利用limma R包（REF）开展翻译组分析。翻译效率（TE）通过下拉富集组分与输入对照样本的mRNA计数比值计算得到。本数据集包含标准化转录本计数、各组间比值与倍数变化计算结果，对应两组比较：转染对照反义寡核苷酸的细胞中，棕榈酸（PA）处理组与BSA处理组的比较；以及转染a-GLU反义寡核苷酸的细胞中，PA处理组与BSA处理组的比较。