EXPERIMENT_TITLE: GSM6278721: MCF10A_NIPBL KD_R3_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: MCF10A cells (3 mill cells per condition) were fixed with 2% formaldehyde (Sigma, #252549) in PBS with 10% FBS for 10 min. Formaldehyde was quenched with 300 mM of glycine at RT for 5 minutes. Hi-C experiments were performed with Arima-HiC Kit (A510008) following manufacturer's instructions. Libraries were prepared with Swift Biosciences Accel-NGS 2S Library Kit (Cat# 21024) and amplified with KAPA Library Amplification Kit (KAPA Cat# KK2620). For all the libraries, 8-9 PCR cycles were used for amplification and they were then sequenced on an Illumina NextSeq550 (82x43 bp).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Cohesin mediates sister chromatid cohesion and organizes the genome through the formation of chromatin loops. Two versions of the complex carrying either STAG1 or STAG2 show overlapping and specific functions and both are required to fulfill embryonic development. Cohesin-STAG1 displays longer residence time on chromatin that depends on CTCF and ESCO1 and establishes longer, long-lived chromatin loops together with CTCF. Cohesin-STAG2 shows a preferential interaction with WAPL and mediates shorter loops involved in tissue-specific transcription independently of CTCF. Here we show that the two variants respond in opposite ways to knock down of NIPBL, the putative cohesin loader that is also essential for loop extrusion. Cohesin-STAG1 levels increase on chromatin under this condition and the complex accumulates further at CTCF positions while cohesin-STAG2 is diminished genome-wide. Our data support a model in which NIPBL is not required for association of cohesin with chromatin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 is preferentially loaded at CTCF sites independently of NIPBL. Nevertheless, loop formation by these chromatin-bound complexes is impaired and gene expression is severely affected, resembling alterations in Cornelia de Lange patients Overall design: In situ Hi-C was performed in mock transfected MCF10A cells (3 replicates) and cells transfected with a single oligonucleotide against NIPBL (2 replicates) or a mix of four oligonucleotides (smart pool, 2 replicates).
SAMPLE_TITLE: MCF10A_NIPBL KD_R3_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "breast"}, {"TAG": "tissue", "VALUE": "breast"}, {"TAG": "cell line", "VALUE": "MCF10A"}, {"TAG": "cell type", "VALUE": "epithelial"}, {"TAG": "treatment", "VALUE": "NIPBL siRNA (smartpool)"}]
Pool_Member_@attributes_sample_title: MCF10A_NIPBL KD_R3_HiC
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