GSM4046018: 4C_p0_cortex_CBSbc_de del_rep2; Mus musculus; OTHER - SRA

SRX6761010: GSM4046018: 4C_p0_cortex_CBSbc_de del_rep2; Mus musculus; OTHER
1 ILLUMINA (HiSeq X Ten) run: 4.7M spots, 1.4G bases, 557.6Mb downloads

Submitted by: NCBI (GEO)

Study: Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [II]

PRJNA562270 • SRP219361 • All experiments • All runs

show Abstracthide Abstract

CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in vivo in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation. Overall design: QHR-4C profiles of P0 wild type (WT) and CBS-deletion mouse cortex were generated by deep sequencing using Illumina HiSeq X-ten CBS deletion mouse lines used in this study (UCSC mouse July 2007(mm9) referance genome): CBSa del chr18:38,001,106-chr18:38,001,113 CBSbcd del chr18:38,011,224-chr18:38,037,839 CBSbc del chr18:38,011,224-chr18:38,013,147 CBSde del chr18:38,036,567-chr18:38,040,872 CBSd del chr18:38,036,568-chr18:38,037,818 CBSb-e del chr18:38011471-chr18:38040871 CBSe del chr18:38,039,423-chr18:38,040,879 Please note that the GSM4046041-GSM4046056 sample records have been moved to GSE211024 on Aug 11, 2022 as per submitter's request.

Sample: 4C_p0_cortex_CBSbc_de del_rep2

SAMN12637193 • SRS5309612 • All experiments • All runs

Organism: Mus musculus

Library:

Instrument: HiSeq X Ten

Strategy: OTHER

Source: GENOMIC

Selection: other

Layout: PAIRED

Construction protocol: P0 mouse brain was dissected, and the tissue from cerebral cortex was digested with 0.013% of collagenase in Neurobasal Medium (Gibco) at 37℃ for 3 min. The collagenase was neutralized by adding excess amount of Neurobasal Medium. Single-cell suspension was made by gentle pipetting, and then filtered through 100-μm cell strainers (BD Biosciences). and the suspension were used for QHR-4C experiments. The single-cell suspension were suspended for crosslinking in 900 ml 2% formaldehyde at room temperature for 10 min. The crosslinking reaction was stopped by adding and mixing with 100 ml of 2M glycine for a final concentration of 200 mM. The fixed cells were spin down at 800 g at 4 °C for 5 min and washed twice by suspending briefly in 1 ml ice-cold PBS. Cells were then permeabilized twice with 200 ml ice-cold 4C permeabilization buffer each for 10 min (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, and 1× protease inhibitors). The cells were then digested overnight at 37 °C with DpnII while shaking at 900 rpm. After inactivation of DpnII at 65 °C for 20 min, proximity ligation was carried out for 24 hr at 16 °C with T4 DNA ligase in 0.1 ml 1 × T4 ligation buffer. The ligated product was then reverse cross-linked and DNA was extracted using phenol-chloroform. Glycogen was added to facilitate DNA precipitation. The precipitated DNA was then dissolved in 50 ml water, and sonicated using the Biorupter system (with low energy setting at a train of 30-second sonication with 30 second intervals for 12 cycles) to obtain DNA fragments ranging from 200 to 600 bp.The captured fragments were linear-amplified using a 5' biotin-tagged primer complementary to the viewpoint fragment in 100ul of PCR system for a total of 60 cycles. This primer should be neither too close to the DpnII site to facilitate the nested PCR at the final amplification step, nor too far away from the DpnII site to maximize the product amount. The amplification products were denatured by incubating at 95 °C for 5 min, and immediately chilled on ice to obtain ssDNA. The ssDNA was then enriched and purified with Streptavidin Magnetic Beads (Invitrogen) according to the manufacturer's instructions. The ssDNA on-beads was then ligated in 15 µl with 0.1 µM of adapters (Supplementary Table 1) at 16 °C for 24 hr. The adapters were generated by annealing two complementary primers matching Illumina P7 sequences. After ligation, free adapters were removed by washing the beads twice with the B/W Buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, pH 7.5). Finally, the QHR-4C libraries were generated by one-step PCR amplification (94 °C, 2 min; 94 °C, 10 sec, 60 °C, 15 sec, 72 °C, 1 min for 19 cycles; and a final extension at 72 °C, 5 min) with captured ssDNA on beads as the template and a pair of PCR primers. The forward primer matches the Illumina P5 and the viewpoint sequence adjacent to the viewpoint DpnII site with barcodes, and the reverse primer matches Illumina P7 with indexes. The PCR products were purified with a PCR purification kit (Qiagen).

Experiment attributes:

GEO Accession: GSM4046018

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 4.7M spots, 1.4G bases, 557.6Mb

Run	# of Spots	# of Bases	Size	Published
SRR10023972	4,715,006	1.4G	557.6Mb	2020-03-02

ID:: 8905546

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