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Series GSE135621 Query DataSets for GSE135621
Status Public on Jul 05, 2020
Title A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of the zygotic genome activation programm (10X Genomics CRISPRa screen dataset)
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics and applied this method to systematically probe candidate regulators of ZGA-like transcription. We screened 230 epigenetic and transcriptional factors by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq), we generated approximately 200,000 single-cell transcriptomes of CRISPRa-perturbed cells, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using multi-omics factor analysis (MOFA) of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. We further validated nine candidate genes by arrayed CRISPRa analysed by bulk transcriptomics, which demonstrates that the combination of CRISPRa with scRNA-seq is a powerful and valid approach to identify regulators of ZGA-like transcription. Additional cDNA overexpression assays for three top hits, the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, confirmed these factors as ZGA-like regulators by alternative methods. Supporting these findings, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes and functional experiments revealed that Smarca5’s regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.
 
Overall design SAM mESCs were transduced with a lentiviral library of 475 short-guide RNAs (sgRNAs) targeting maternal epigenetic factors, in triplicates at a <0.1 multiplicity-of-infection (MOI) , and following selection and expansion of the pool of transduced cells, single-cell transcriptomes were generated using 10X 3’ scRNA-seq and their corresponding sgRNAs further amplified with a specific amplification protocol (Hill et al. 2018). Each transduction replicate was loaded across a full 10X chip Chromium Controller (8 lanes), with 20,000 cells per lane. Samples names are labelled with the replicate number 1-3 and 10X lane 1-8 (e.g: Replicate1_1 refers to transduction replicate 1 run in 10X lane 1). Amplicon sgRNA libraries were performed for each full length 10X cDNA sample (8 per replicate) and are referred to with the replicate number 1-3 and 10X lane 1-8 (e.g. amplicon_sgRNA_1_1 refers to the amplicon sgRNA libraries made on the 10X full length cDNA of sample1_1).
 
Contributor(s) Alda-Catalinas C, Bredikhin D, Hernando-Herraez I, Santos F, Kubinyecz O, Eckersley-Maslin MA, Stegle O, Reik W
Citation(s) 32634384
Submission date Aug 09, 2019
Last update date Aug 03, 2020
Contact name Felix Krueger
E-mail(s) felix.krueger@babraham.ac.uk
Organization name The Babraham Institute
Department Bioinformatics
Street address Babraham Hall
City Cambridge
ZIP/Postal code CB22 3AT
Country United Kingdom
 
Platforms (2)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
GPL21103 Illumina HiSeq 4000 (Mus musculus)
Samples (27)
GSM4023607 amplicon_sgRNA_1
GSM4023608 amplicon_sgRNA_2
GSM4023609 amplicon_sgRNA_3
This SubSeries is part of SuperSeries:
GSE135622 A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation
Relations
BioProject PRJNA559476
SRA SRP217912

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
GSE135621_RAW.tar 4.0 Gb (http)(custom) TAR (of MTX, TSV)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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