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| Status |
Public on Jun 18, 2020 |
| Title |
A549_Input_r3 |
| Sample type |
SRA |
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| Source name |
Adenocarcinomic human alveolar basal epithelial cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: lung
treatment: 0.5% O2 16h
type of sample: Input
chip antibody: none
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| Growth protocol |
cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 5ml of 100 U/ml Penicillin-Streptomycin (Pen Strep, Life Technologies) and 5ml of L-Glutamine 200mM.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated in hypoxic conditions for 16 hours. Cultured cells were subsequently immediately fixed by adding 1% Formaldehyde (16% Formaldehyde (w/v), Methanol-free, Thermo Scientific) directly in the medium and incubating for 8 minutes. Fixed cells were incubated with 150 mM of glycine for 5 min to revert the cross-links, washed twice with ice-cold PBS 0.5% Triton-X100, scraped and collected by centrifugation (1000 ×G 5min at 4°C). The pellet was resuspended in 1400 µL of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton-X100, 0.5% Sodium deoxycholate, 1% SDS, 1% protease inhibitors) and transferred in a new eppendorf tube. The chromatin was sonicated for 3 min by using a Branson 250 Digital Sonifier with 0.7 s 'On' and 1.3 s 'Off' pulses at 40% power amplitude, yielding a size of 100 to 500 bp. The sample was kept ice-cold at all times during the sonication. The samples were centrifuged (10 min at 16000 ×G at 4°C) and the supernatant were transferred in a new eppendorf tube. The protein concentration was assessed using a BCA assay. Fifty µL of shared chromatin was used as “input” and 1.4 µg of primary ARNT/HIF-1β monoclonal antibody (NB100-124, Novus) per 1 mg of protein was added to the remainder of the chromatin, and incubated overnight at 4°C in a rotator. Pierce Protein A/G Magnetic Beads (Life Technologies) were added to the samples in a volume that is 4X the volume of the primary Ab and incubated at 4°C for at least 5 hours. A/G Magnetic Beads were collected and the samples were washed 5 times with the washing buffer (50 mM Tris-HCl, 200 mM LiCl, 2 mM EDTA, pH 8, 1% Triton, 0.5% Sodium deoxycholate, 0.1% SDS, 1% protease inhibitors), and twice with TE buffer. The A/G magnetic beads were resuspended in 50 µL of TE buffer, and 1.5 µL of RNAse A (200 units, NEB, Ipswich, MA, USA) were added to the A/G beads samples and to the input, incubated for 10 minutes at 37°C. After addition of 1.5 µL of proteinase K (200 units) and overnight incubation at 65°C, the DNA was purified using 1.8´ volume of Agencourt AMPure XP (Beckman Coulter) according to the manufactory instructions
One µg of input and all of the immunoprecipitated DNA were converted into sequencing libraries using the NEBNext Ultra DNA library prep kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
A549
A549.macs.peaks.txt
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| Data processing |
FastQ files were mapped to the reference genome (human:build GRCh37/hg19; mouse:mm10) using bowtie (version 0.12.8; settings -v 2 -m 1 -p --best -S).
Peaks were called by model-based analysis of ChIP-sequencing (MACS) using pooled data for each cell line and using the correspondent input as background set.
Genome_build: hg19; mm10
Supplementary_files_format_and_content: MACS called peaks for each cell line
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| Submission date |
Sep 12, 2018 |
| Last update date |
Jun 18, 2020 |
| Contact name |
Jieyi Xiong |
| Organization name |
VIB-KULeuven Center for Cancer Biology
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| Street address |
Herestraat 49, box 912
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE85352 |
DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumour immunotolerance [ChIPSeq] |
| GSE85356 |
DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumour immunotolerance |
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| Relations |
| BioSample |
SAMN10037531 |
| SRA |
SRX4672368 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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