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Series GSE195992 Query DataSets for GSE195992
Status Public on Feb 04, 2022
Title Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes
Organism Homo sapiens
Experiment type Other
Summary Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing . Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells. 
 
Overall design We performed mutagenesis of the terminal repeat region of the piggyBac transposon to identify sites that could accommodate or serve as a barcode for self-reporting transposon calling card expeirments. We identified 4 mutable nucleotides and used these sites as barcodes. We constructed barcoded self-reporting transposons encoding either puromycin resistance or tdTomato. We then used these barcoded constructs to identify binding sites for four transcription factors involved in cell fate decisions.
 
Contributor(s) Lalli MA, Mitra RD
Citation(s) 36062164
Submission date Feb 02, 2022
Last update date Sep 15, 2022
Contact name Matthew A Lalli
Organization name Icahn School of Medicine at Mount Sinai
Department Psychiatry
Street address 1 Gustave L. Levy Pl
City New York
State/province NY
ZIP/Postal code 10029
Country USA
 
Platforms (2)
GPL15520 Illumina MiSeq (Homo sapiens)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (46)
GSM5857631 ASCL1 mix tdTomato BC SRT Rep1
GSM5857632 ASCL1 mix tdTomato BC SRT Rep2
GSM5857633 ASCL1 mix tdTomato BC SRT Rep3
Relations
BioProject PRJNA802849

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Supplementary file Size Download File type/resource
GSE195992_RAW.tar 58.0 Mb (http)(custom) TAR (of TXT)
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Raw data are available in SRA
Processed data provided as supplementary file

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