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Series GSE61189 Query DataSets for GSE61189
Status Public on Nov 05, 2014
Title Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification
Organism Mus musculus
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Summary The aim of this study was to determine the genomic binding sites of an HA-tagged Transcription Activator-Like Effector (TALE) fused to a VP64 domain with a DNA binding domain designed to bind the sequence GGGCGCTTCCTGTTTTCTCA (found in the PU.1-14kb enhancer element in mouse and human genome), termed HA-T-VP64-PU.1-14, in the 416B mouse myeloid progenitor cell line.
Overall design Inducible T-VP64-PU.1-14 transgene contained within the piggyBac vector was stably integrated into 416B cells by transfection along with a constituteively expressed rtTA plasmid (pCAG-rtTA-piggyBac) and a piggyBac transposase. 416Bs carrying stably integrated transgenes were FACS sorted based on their ability to expressed the transgene and expanded before HA-T-VP64-PU.1-14 expression was induced for 48 hours by addition of dox before cells were fixed with 1% formaldehyde for 10 mins. Chromatin was isolated, sonicated for 7 mins (30 sec on, 30 sec off), and an anti-HA antibody used to pull down the HA-T-VP64-PU.1-14 after a pre-clearing step. Chromatin was washed, de-crosslinked, amplified, size selected by gel purification and sequenced. As a control, untransfected 416B cells were similarly ChIP'd.
Contributor(s) Wilkinson AC
Citation(s) 25252941
Submission date Sep 08, 2014
Last update date May 15, 2019
Contact name Bertie Gottgens
Organization name University of Cambridge
Street address Jeffrey Cheah Biomedical Centre
City Cambridge
ZIP/Postal code CB2 0AW
Country United Kingdom
Platforms (1)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
Samples (2)
GSM1499158 416B_Ctrl_HA
GSM1499159 416B_HA-T-VP64-PU.1-14_HA
BioProject PRJNA260492
SRA SRP046309

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Supplementary file Size Download File type/resource
GSE61189_RAW.tar 300.1 Mb (http)(custom) TAR (of BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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