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Series GSE226161 Query DataSets for GSE226161
Status Public on Nov 05, 2023
Title Profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing
Organisms Homo sapiens; Mus musculus
Experiment type Expression profiling by high throughput sequencing
Other
Summary RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA substrates are still lacking, especially when the interaction is dynamic or materials are limited. Here we present an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq), which relies on in-situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet cross-linking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic binding of RBPs over a short period of time, as demonstrated by the discovery of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 min.
 
Overall design We first developed an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq) to identify PTBP1 binding sites in HepG2 cells. To validate the PTBP1 binding sites identified by ARTR-seq, we performed ARTR-seq for PTBP1 knocked down HepG2 cells. To determine the efficiency of ARTR-seq for small cell number inputs, we constructed ARTR-seq libraries using different numbers of HepG2. We next constructed ARTR-seq libraries to identify the binding sites for several RBPs, including RBFOX2 and HNRNPC in HepG2 cells, and YTHDF1, YTHDF2 and YTHDC1 in HeLa cells. We then generated ARTR-seq libraries in a section of OCT-embedded E11 mouse embryos to identify the RBFOX2 binding sites. To monitor the dynamic binding of G3BP1 during the stress granules assembly, we performed ARTR-seq for the HeLa cells treated with sodium arsenite for various time intervals.
 
Contributor(s) Chen Y, Xiao Y, He C
Citation(s) 38200227
Submission date Feb 27, 2023
Last update date Jan 11, 2024
Contact name Yanming Chen
E-mail(s) yanmingchen@uchicago.edu
Organization name The University of Chicago
Street address 929 E 57th Street
City Chicago
ZIP/Postal code 60637
Country USA
 
Platforms (3)
GPL21626 NextSeq 550 (Mus musculus)
GPL21697 NextSeq 550 (Homo sapiens)
GPL30173 NextSeq 2000 (Homo sapiens)
Samples (89)
GSM7065957 A.HepG2.40k.Input.rep1
GSM7065958 A.HepG2.40k.Input.rep2
GSM7065959 A.HepG2.40k.Input.rep3
Relations
BioProject PRJNA939112

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE226161_RAW.tar 1.4 Gb (http)(custom) TAR (of BW, TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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