RAG-seq:NSR-primed and Transposase Tagmentation-mediated Strand-specific Total RNA Sequencing in Single Cells  

作　　者：Ping Xu Zhiheng Yuan Xiaohua Lu Peng Zhou Ding Qiu Zhenghao Qiao Zhongcheng Zhou Li Guan Yongkang Jia Xuan He Ling Sun Youzhong Wan Ming Wang Yang Yu 

机构地区：[1]China-Japan Union Hospital of Jilin University,Jilin University,Changchun 130033,China [2]School of Life Sciences,Jilin University,Changchun 130012,China [3]Guangzhou Women and Children's Medical Center,Guangzhou Medical University,Guangzhou 510623,China [4]Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China [5]Center for Reproductive Medicine,Guangzhou Women and Children's Medical Center,Guangzhou Medical University,Guangzhou 510623,China

出　　处：《Genomics, Proteomics & Bioinformatics》2024年第5期73-84,共12页基因组蛋白质组与生物信息学报(英文版)

基　　金：supported in part by grants from the National Natural Science Foundation of China(Grant Nos.81921003 and 32170605 to YY,Grant No.32100473 to LG);the Ministry of Science and Technology of China(Grant Nos.2019YFA0508903 and 2017YFA0504200 to YY);the Jilin Province Health Research Special Fund for Outstanding Talented Person(Grant No.2023SCZ58 to YW);YY was additionally supported by the start-up fund from Guangzhou Women and Children's Medical Center.

摘　　要：Single-cell RNA sequencing(scRNA-seq)has transformed our understanding of cellular diversity with unprecedented resolution.However,many current methods are limited in capturing full-length transcripts and discerning strand orientation.Here,we present RAG-seq,an innovative strand-specific total RNA sequencing technique that combines not-so-random(NSR)primers with Tn5 transposase-mediated tagmentation.RAG-seq overcomes previous limitations by delivering comprehensive transcript coverage and maintaining strand orientation,which are essential for accurate quantification of overlapping genes and detection of antisense transcripts.Through optimized reverse transcription with oligo-dT primers,rRNA depletion via Depletion of Abundant Sequences by Hybridization(DASH),and linear amplification,RAG-seq enhances sensitivity and reproducibility,especially for low-input samples and single cells.Application to mouse oocytes and early embryos highlights RAG-seq's superior performance in identifying stage-specific antisense transcripts,shedding light on their regulatory roles during early development.This advancement represents a significant leap in transcriptome analysis within complex biological contexts.

关 键 词：Antisense transcript FULL-LENGTH Mouse early embryonic development Single-cell RNA sequencing Strand-specific 

分 类 号：R73[医药卫生—肿瘤]