EXPERIMENT_TITLE: GSM5070285: C-TALE_control_rep3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: C-TALE was performed as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan). For ChIP-seq experiments, fixed chromatin pellets were resuspended in 600 uL of ice cold RIPA buffer supplemented with protease inhibitors. Chromatin was sheared on ice with a VirSonic 100 cell disrupter with 10 30-sec pulses on “15” power setting, separated with 3 min periods of recovery. Input DNA was isolated from 1/10 aliquots of sonicated samples. Sheared chromatin from approximately 1 mln of cells and 1 ug of antibodies (against either CTCF, Smc3, or Rad21) were used in each immunoprecipitation reaction. Chromatin immunoprecipitation was performed as described in Abcam X-ChIP manual (https://www.abcam.com/protocols/cross-linking-chromatin-immunoprecipitation-x-chip-protocol) with minor modifications. 25 ul of protein A/G magnetic beads (Thermo Scientific, 26162) was used per reaction instead of agarose beads, thus a magnet was used instead of a centrifuge to reclaim beads. Beads were blocked with 1% BSA solution in RIPA overnight, while chromatin was incubated with antibodies. BSA-blocked beads were mixed with antibody-bound chromatin for 6 h. Immunoprecipitated DNA was separated from beads by overnight treatment with proteinase K (Thermo Scientific, EO0491) in PBS supplemented with 1% SDS at 65C. Solution was then cleared from beads with a magnet and DNA was isolated with standard phenol-chloroform extraction and ethanol precipitation. Sequencing libraries from 3C-seq material, immunoprecipitated DNA and ChIP inputs were generated as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Cohesin is a key organizer of chromatin folding in eukaryotic cells. Two basic activities of this ring-shaped protein complex are maintenance of sister chromatid cohesion and establishment of long-range DNA-DNA interactions through the process of loop extrusion. Though basic principles of both cohesion and loop extrusion have been described we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed stability of cohesin-dependent loops and cohesin association with chromatin in high ionic strength conditions in G1-synchronised HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions which correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. As topologically-engaged cohesin is considered to be salt-resistant, our data corroborate a non-topological model of loop extrusion. Overall design: We used C-TALE and ChIP-seq to assess sensitivity of cohesin-chromatin association to high salt treatment in HeLa cells
SAMPLE_TITLE: C-TALE_control_rep3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HeLa cells"}, {"TAG": "cell line", "VALUE": "HeLa"}, {"TAG": "cell type", "VALUE": "cervical carcinoma cells"}, {"TAG": "treatment", "VALUE": "isotonic buffer"}]
Pool_Member_@attributes_sample_title: C-TALE_control_rep3
RUN_SET_RUN_Pool_Member_@attributes_sample_title: C-TALE_control_rep3
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR13658192.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos4/sra-pub-zq-1/SRR013/13658/SRR13658192/SRR13658192.lite.1", "size": "1308458899", "date": "2022-08-10 05:04:58", "md5": "717cb64ed9c1be7a4088cfe8bf4f27bc", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR13658192", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR13658192/SRR13658192", "size": "2197177507", "date": "2021-02-08 19:19:44", "md5": "0bcf03d07a790a3312e922b748d3c95f", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "CTALE_control_3_R2.fastq.gz", "size": "1722410480", "date": "2021-02-08 19:16:23", "md5": "912bd6ebf0d4d9365beec80ad34fc3bf", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "CTALE_control_3_R1.fastq.gz", "size": "1519774124", "date": "2021-02-08 19:17:48", "md5": "3d3ebd44c157dc24444c84c4bbf561dd", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]