EXPERIMENT_TITLE: GSM3514560: HEK_a12pCBS_dCas9-VPR_sorted_HiC_rep3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Mice were sacrificed using CO2 followed by cervical dislocation. The main olfactory epithelium (MOE) was dissected and transferred to ice-cold PBS. The MOE was cut in to small pieces with a razor blade, and then dissociated with a papain dissociation system (Worthington Biochemical). For RNA-seq, dissociated cells were washed once with sort media (PBS + 2% Fetal Bovine Serum) and then resuspended in sort media supplemented with 100 U/mL DNase I (Worthington Biochemical), 4mM MgCl2, and 500ng/mL DAPI (Invitrogen). For Crosslinked-ChIP samples, cells were fixed prior to sorting. For fixation, dissociated cells were resuspended in PBS + 1% methanol-free formaldehyde (Pierce). Cells were fixed at room temperature for 5 minutes, and then fixation was quenched by adding 1/10th volume of 1.25M glycine. Fixed cells were pelleted (500 rcf, 5 minutes, room temperature), washed once with PBS + 2% FBS, and then resuspended in sort media. All cells were passed through a 40uM cell strainer prior to sorting.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Stochastic activation of clustered Protocadherin (Pcdh) a, ß, and ? genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdha alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdha promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdha promoter choice. Overall design: RNA-seq, ChIP-seq, and Hi-C
SAMPLE_TITLE: HEK_a12pCBS_dCas9-VPR_sorted_HiC_rep3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HEK293T cells"}, {"TAG": "cell type", "VALUE": "HEK293T cells"}, {"TAG": "genotype", "VALUE": "HEK293T cells positively transfected with dCas9-VPR targeting the pCBS of Pcdha12"}, {"TAG": "chip antibody", "VALUE": "N/A"}]
Pool_Member_@attributes_sample_title: HEK_a12pCBS_dCas9-VPR_sorted_HiC_rep3
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HEK_a12pCBS_dCas9-VPR_sorted_HiC_rep3
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