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| Status |
Public on Dec 01, 2020 |
| Title |
Control rep2 10h |
| Sample type |
SRA |
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| Source name |
SMC2 ChIP in control HCT116 cells, 10 hours post release from G1/S
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell cycle: G2/M
condition: control
chip antibody: anti-SMC2
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| Treatment protocol |
Cells were synchronised at the G1/S boundary by treatment with 5mg/ml aphidicolin for 24 hours. In addition, some samples were treated with a low dose of aphidicolin (0.4 microM) post release.
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| Growth protocol |
HCT116 (ATCC CCL-247) were cultured in DMEM-F12 supplemented with, 3 mM glutamine, 15 mM HEPES, 10% FBS, penicillin (100 U ml−1), streptomycin (100 μg ml−1) and phenol red (8.1 mg l−1).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Synchronised HCT116 cells were cross-linked first with 1 mM disuccinimidyl suberate (DSS, sigma) for 40 minutes and then with 1% formaldehyde for 20 minutes at room temperature, and then incubate in 200 mM glycine for 5 min to quench reactive DSS and formaldehyde. Cells were scraped, flash-frozen and stored at -20 °C. Cell pellets were lysed in 100 µl SDS Lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM EDTA, and 1% SDS) and added ChIP dilution buffer (50 mM Tris-HCl [pH7.5], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, and protease inhibitor cocktail complete EDTA-free (Roche)) before sonication (Soni-prep sonicator for 20 cycles [40 seconds on, 20 seconds off]). Samples were then spun at 13000 rpm for 15 minutes at 4 °C, supernatant was collected and 10% of the supernatant volume was saved as an input. Immunoprecipitation was performed with Dynabeads protein G beads (Invitogen) coupled to an anti-SMC2 antibody provided by Kumiko Samejima. Samples were mixed with beads and incubated at 4 °C for 4 hours with rotation and washed sequentially with RIPA-150 mM NaCl and RIPA-500 mM NaCl (50 mM Tris-HCl [p8.0H], 150/500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and 0.1% sodium deoxycholate) and TE, then added direct elution buffer (10 mM Tris-HCl [pH8.0], 300 mM NaCl, 5 mM EDTA, and 0.5% SDS) before overnight at 65 °C to reverse cross-linking. Samples were treated with 5/200 units/ml RNaseA/T1 (Ambion) and 250 µg/ml proteinase K. DNA was extracted from the input and ChIP samples via phenol/chloroform precipitation.
Libraries were prepared with a NEBNext® Ultra II™ DNA Library Prep Kit for Illumina (NEB), according to manufacturer’s instructions and sample uniquely barcoded using NEBNext Multiplex oligo for Illumina (NEB). Samples were quantified using Qubit using a High Sensitivity reagents. Libraries were analysed by Edinburgh Genomics at the University of Edinburgh, UK, who performed quality control and sequencing using an Illumina NovaSeq, 100 base paired-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
SMC2 ChIP in control HCT116 cells, 10 hours post release from G1/S, replciate 2
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| Data processing |
Fastq files were aligned to hg 19 using Bowtie 2
PCR duplicate removal, file sorting and indexing was performed with Samtools 1.2
Bam files were generated and read density in 1000 bp windows across the genome were calculated using the "multicov" option in Bedtools 2.25
Normalised FPKM values were generated for each window in R
FPKM values for genomic input for the corresponding cell cycle stage was subtracted
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph files containing FPKM values normalised for genomic input in 1kb windows only for chromosomes 2 and 4. Genome-wide data were not produced as part of this study as the focus was on chr 2 and 4.
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| Submission date |
Dec 17, 2018 |
| Last update date |
Dec 02, 2020 |
| Contact name |
Lora Boteva |
| E-mail(s) |
[email protected]
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| Phone |
+44 (0) 131 651 8551
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| Organization name |
University of Edinburgh
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| Department |
Institute of Genetics and Molecular Medicine
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| Lab |
Gilbert Lab
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| Street address |
Crewe Road
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| City |
Edinburgh |
| State/province |
City of Edinburgh, Midlothian |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE123918 |
ChIP of SMC2 at defined cell cycle stages |
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| Relations |
| BioSample |
SAMN10601795 |
| SRA |
SRX5139488 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3516866_control2_10h_chr2_cor.bedGraph.gz |
15.1 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM3516866_control2_10h_chr4_cor.bedGraph.gz |
14.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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