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| Status |
Public on Jul 05, 2016 |
| Title |
REH H3K4me3 [ChIP-Seq] |
| Sample type |
SRA |
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| Source name |
REH cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: ALL leukemia cell line REH
antibody: H3K4me3 (Abcam, catalog# 8580)
replicate: 1
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| Treatment protocol |
Cells in basal conditions
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| Growth protocol |
Cells were cultured in RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) suppelemented with 2 mM L-glut, 100 U penicillin, 100 ug/ml streptomycin and 10% Tet System Approved FBS (Clontech, Mountain View, CA, USA).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
40 million cells were crosslinked with 1% formaldehyde for 10-15 mins. The reactions were quenched by adding glycine to a final concentration of 125 mM, and the cells were centrifuged and washed twice with ice-cold PBS. For each ChIP 5 million cells was used. Nuclei was extracted by washing cell pellet twice with 1 ml of MNase buffer (10mM Tris ph 7,4, 10mM NaCl, 5mM MgCl2, 0,5% IGEPAL CA-630 [Sigma], 1x protease inhibitor cocktail [PIC, Roche], 1 mM PMSF [Thermofisher]). Nuclei were spun down (1500 x g, +4 °C, 5 min) and suspended into 90µl of MNase buffer supplemented with 5mM CaCl2 and 0.1% Triton-X. Different amounts of MNase (0.5-20U; #88216, Thermofisher) was added to the nuclei in 10 µl volume and incubated at 37°C for 10 mins. To stop the reaction, 100 µl of 2xLysis buffer was added to the reaction (1% SDS, 40mM EDTA, 100mM Tris-HCl pH 8.1) and samples were sonicated using Bioruptor (Diagenode) for 5 cycles (30 s – 30 s) to break the nuclei. The lysate was cleared by centrifugation and supernatant was diluted 5 x with dilution buffer (for H3K4me3; 20mM Trix-HCl pH 7.4, 100mM NaCl, 2mM EDTA, 0.5% TritonX, PIC). The diluted lysate was pre-cleared by rotating for 2 h at 4°C with 60 µl 80% CL-4B sepharose slurry (GE Healthcare; Before use, sepharose was washed twice with TE buffer, blocked for 1h min at room temperature with 0.5% BSA and 20 µg/ml glycogen in 1 ml TE buffer, washed twice with TE and brought up to the original volume with TE). The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 3-5 µg antibody overnight at 4°C. Antibody H3K4me3 (ab8580) was purchased from Abcam. The Ab was captured using 25 µl blocked Protein G Sepharose® 4 Fast Flow (GE Healthcare, sepharose was blocked rorating overnight at 4°C) and rotating the sample for 2 h at 4°C. The beads were pelleted (1 min, 1000× g, 4°C), the supernatant discarded, and were washed three times with wash buffer I (20 mM Tris/HCl pH 7.4@20°C, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), twice with buffer II (20 mM Tris/HCl pH 7.4@20°C, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA) and buffer III (10 mM Tris/HCl pH 7.4@20°C, 250 mM LiCl, 1% IGEPAL CA-630, 1% Na-deoxycholate, 1 mM EDTA), once with TE+0.2% TritonX and twice with TE.
Immunoprecipitated chromatin was eluted twice with 100 µl elution buffer (TE, 1% SDS). The NaCl concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C. The samples were sequentially incubated at 37°C for 2 h each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adaptor ligation as previously described (Heinz 2010; MolCell) using barcoded adapters (NextFlex, Bioo Scientific). Between the reactions, the DNA was purified using Sera-Mag SpeedBeads (Thermo Scientific). Libraries were PCR-amplified for 15-16 cycles and size selected for 230-350bp fragments by gel extraction.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Immunoprecipitated DNA
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| Data processing |
Libraries were single-end sequenced on a Hi-Seq 2000 (Illumina) for 51 cycles, according to the manufacturer’s instructions.
Sequence reads were quality filtered with the FASTX software (-q 10 -p 97) and read stacks were collapsed.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie version 0.12.9 (-v 2 -k 3 -m 1 --best)
Bedgraphs were generated using the HOMER software. The Pol2 bedgraphs were generated at 50bp resolution for change point analysis. Data analysis was performed using HOMER and custom scripts http://biowhat.ucsd.edu/homer/
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph ChIP-seq signal files
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| Submission date |
May 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Merja Heinäniemi |
| Organization name |
university of eastern finland
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| Street address |
Yliopistonranta 1
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| City |
Kuopio |
| ZIP/Postal code |
70210 |
| Country |
Finland |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE67540 |
RNA polymerase in pre-B-ALL cell lines |
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| Relations |
| BioSample |
SAMN05150244 |
| SRA |
SRX1776295 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2166075_REH_H3K4me3_default_hg19.ucsc.bedGraph.gz |
38.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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