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Series GSE137925 Query DataSets for GSE137925
Status Public on Apr 25, 2021
Title Global Profiling of RNA-Binding Protein Target Sites by LACE-seq
Organisms Homo sapiens; Mus musculus
Experiment type Other
Expression profiling by high throughput sequencing
Summary RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of an RBP in those rare cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of cDNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4, and PTBP1, in mature oocytes. Unexpectedly, transcriptomic and proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes could fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP binding sites in a few cells opens the door for studying the roles of RBPs in embryonic development and reproductive diseases.
Overall design We first developed a linear amplification of cDNA ends and sequencing (LACE-seq) methodology to identify RBP targets at or near the single-cell level. Next, we determined the binding sites for several RBPs, including Argonaute 2 (Ago2), MILI, Ddx4, and PTBP1 (also known as PTB) in single or dozens of fully-grown mouse oocytes. Two biological repeats, labeled as Rep 1 and Rep 2, were generated for each sample. For measuring Ago2 regulated genes in fully-grown oocytes, we also performed single-cell RNA-seq analysis on five control and Ago2-null oocytes. To identify endo-siRNAs loaded into Ago2, we performed single-cell CAS-seq using Ago2 immunoprecipitated small RNAs from MII oocyte and labeled as Ago2_IP_Oocyte. To evaluate the specificity of LACE-seq, we performed Ago2 LACE-seq in oocytes from Ago2 conditional knockout (cKO) mice and PTBP1 LACE-seq in K562 cells treated with PTBP1 shRNA.
RNA-seq analysis of Meiosis II oocytes from Ago2 cKO (Ago2 loxP/loxP,Zp3-cre) and Control(Ago2 loxP/+) female mice.
Contributor(s) Su R, Fan L, Cao C, Wang L, Xue Y
Citation(s) 34108658
Submission date Sep 24, 2019
Last update date Jun 24, 2021
Contact name Yuanchao Xue
Organization name Chinese Academy of Sciences
Department Institute of Biophysics
Lab Key Laboratory of RNA Biology
Street address Datun Road #15
City Beijing
ZIP/Postal code 100101
Country China
Platforms (5)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (55)
GSM4094519 PTB_10000HeLaCells_rep1
GSM4094520 PTB_10000HeLaCells_rep2
GSM4094521 PTB_1000HeLaCells_rep1
BioProject PRJNA573894
SRA SRP223080

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Supplementary file Size Download File type/resource
GSE137925_Ago2.50_oocytes.bigwig 14.6 Mb (ftp)(http) BIGWIG
GSE137925_Ago2.50_oocytes.withoutIVT.bigwig 6.8 Mb (ftp)(http) BIGWIG
GSE137925_Ago2.KO_oocytes.bigwig 13.7 Mb (ftp)(http) BIGWIG
GSE137925_Ago2.WT_oocytes.bigwig 25.4 Mb (ftp)(http) BIGWIG
GSE137925_PTBP1.100K.HeLa.bigwig 251.0 Mb (ftp)(http) BIGWIG
GSE137925_PTBP1.100K.K562.withoutIVT.bigwig 252.2 Mb (ftp)(http) BIGWIG
GSE137925_PTBP1.100K.K562_KD.bigwig 43.0 Mb (ftp)(http) BIGWIG
GSE137925_PTBP1.100K.K562_WT.bigwig 119.1 Mb (ftp)(http) BIGWIG
GSE137925_PTBP1.10M.K562.withoutIVT.bigwig 128.5 Mb (ftp)(http) BIGWIG
GSE137925_RAW.tar 458.1 Mb (http)(custom) TAR (of BIGWIG, BW, TXT)
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Raw data are available in SRA
Processed data provided as supplementary file
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