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 s...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.展开更多
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)+1 种基金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.
文摘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.
