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Series GSE81750 Query DataSets for GSE81750
Status Public on Apr 13, 2017
Title Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of 15 SEs, 71 HSs, 241 sgRNAs
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary Transcriptional enhancers orchestrate cell-type specific gene expression programs critical to eukaryotic development, physiology, and disease. However, despite the large number of enhancers now identified, only a small number have been functionally assessed. Here, we develop MOsaic Single-cell Analysis by Indexed CRISPR Sequencing (Mosaic-seq), a method that measures one direct phenotype of enhancer repression: change of the transcriptome, at the single cell level. Using dCas9-KRAB to suppress enhancer function, we first implement a multiplexed system to allow the simultaneous measurement of the transcriptome and detection of sgRNAs by single cell RNA sequencing. We validate this approach by targeting the HS2 enhancer in the well-studied beta-globin locus. Next, through computational simulation, we demonstrate strategies to robustly detect changes in gene expression in these single cell measurements. Finally, we use Mosaic-seq to target 71 hypersensitive regions belonging to 15 super-enhancers in K562 cells by utilizing a lentiviral library containing 241 unique-barcoded sgRNAs. Our results demonstrate that Mosaic-seq is a reliable approach to study enhancer function in single cells in a high-throughput manner.
 
Overall design The overall design consists of RNA sequencing in bulk and in single K562 cells. As a control, we performed single-cell RNA sequencing of K562 cells expressing dCas9-KRAB and a control sgRNA targeting the non-expressed HSBP1 gene in two biological replicates. As a pilot project, we performed single cell RNA-Seq of dCas9-KRAB K562 cells after pooled infection of a library of 10 sgRNA viruses targeting promoters and enhancers in the beta-globin locus. As controls for this experiment, we individually infected these 10 sgRNAs and either performed 1) bulk RNA sequencing or 2) single cell RNA sequencing after pooling the separately infected cells. Finally, we scaled this single-cell approach to measure transcriptome changes for a library of 241 sgRNAs spanning 71 hypersensitive sites from 15 super-enhancers in K562 cells.
 
Contributor(s) Hon G, Xie S
Citation(s) 28416141
Submission date May 23, 2016
Last update date May 15, 2019
Contact name Gary Chung Hon
Organization name UT Southwestern
Department OB/GYN
Street address 5323 Harry Hines Blvd.
City Dallas
State/province TX
ZIP/Postal code 75390
Country USA
 
Platforms (1)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (60)
GSM2175064 K562 dCas9-KRAB, Lenti-sgRNA set B pooled infection, batch 5
GSM2175065 K562 dCas9-KRAB, Lenti-sgRNA set B pooled infection, batch 6
GSM2175066 K562 dCas9-KRAB, Lenti-sgRNA set C pooled infection, batch 8
This SubSeries is part of SuperSeries:
GSE81884 Multiplexed engineering and analysis of endogenous enhancer activity in single cells
Relations
BioProject PRJNA322844
SRA SRP075707

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SOFT formatted family file(s) SOFTHelp
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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE81750_Mosaic-Seq_15SE_71HS_241sgRNA_K562.full_matrix.final.txt.gz 327.0 Mb (ftp)(http) TXT
GSE81750_Mosaic-Seq_15SE_71HS_241sgRNA_K562.sgRNA_to_sgRNA_barcode.txt.gz 6.0 Kb (ftp)(http) TXT
GSE81750_RAW.tar 276.7 Mb (http)(custom) TAR (of TXT)
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record
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