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| Status |
Public on Mar 03, 2023 |
| Title |
CREB3L2-CREB3L2 homodimers (Replicate 2) V5 immunoprecipitation |
| Sample type |
SRA |
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| Source name |
HEK293 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human embryonic kidney cells
dna transfection: HA-CREB3L2-FRB + V5-CREB3L2-FKBP
ChIP: V5 immunoprecipitation
chip antibody-conjugated beads: NC0777490, MBL International - magnetic beads pre-conjugated with anti-V5 antibody
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| Treatment protocol |
DNA constructs were transiently transfected using Lipofectamine 3000 (Thermo Fisher) and amounts of DNA delivery were optimized to achieve comparable expression levels between the different homodimer and heterodimer configurations. While lipofectamine complexes were incubating, a complete media change was performed, and A/C heterodimerizer (Takara) was added at 500 nM at this point. ChIPmera system was incubated for 24 hours.
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| Growth protocol |
HEK293T cells (#CRL-3216, ATCC), plated in 150 mm dishes (CLS430599, Corning), were maintained in DMEM supplemented with 10% fetal bovine serum plus antibiotics (50 U/ml penicillin-streptomycin).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Twenty-four hours post-transfection, we proceeded by crosslinking protein-DNA contacts with 1% (v/v) formaldehyde (#28908, Thermo Fisher) for 10 minutes at room temperature. After quenching cross-linking reaction with glycine, cells were washed twice with ice-cold PBS, harvested by scrapping in PBS with protease inhibitors, and centrifuged at 2,000 g and 4ºC, as per manufacturer’s instructions (SimpleChIP Plus Kit [#9005], Cell Signaling Technology). Chromatin fragments (mainly 1-3 nucleosomes in size) were obtained by incubation with micrococcal nuclease (3.5 ul [equivalent to 7,000 gel units] in 200 ul; M0247S, NEB) for 45 minutes in a 37ºC-water bath. Nuclear membranes were subsequently broken up by three rounds of 20-second, 15% amplitude pulses using a Sonic Dismembrator Model 500 (Fisher Scientific), and lysates clarified by centrifugation. Adequate digestion was assessed by agarose gel electrophoresis. For each condition, digested chromatin was split into two tubes and immunoprecipitated overnight at 4ºC with end-over-end rotation using pre-washed magnetic beads conjugated with anti-HA- or anti-V5 antibodies (anti-HA beads: PI88836, Thermo Fisher; anti-V5 beads: NC0777490, MBL International); 35 ul of anti-HA beads and 25 ul of anti-V5 beads were utilized per immunoprecipitation. Beads were captured on a magnetic stand and washed with low- and high-salt buffers, as directed. Elution was performed at 65ºC and 1,200 rpm for 30 minutes using a thermomixer, protein-DNA cross-links reversed by treatment with Proteinase K for 2 hours at 65ºC, and DNA column-purified. A representative 2% input sample was prepared by combining chromatin from the different backgrounds.
ChIP-seq library preparation and sequencing reactions were conducted at GENEWIZ, Inc., as described above. A total of 24 samples were submitted for analysis, comprising parallel HA and V5 immunoprecipitations, from 2 independent replicates. The sequencing libraries were multiplexed and clustered on two lanes of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Replicate 2
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| Data processing |
ChIP-seq sequencing data were processed and analyzed within the Galaxy web platform. First, library adapters and low quality reads were removed using Trim Galore (version 0.6.3) with the following settings: phred quality score threshold = 20, overlap with adapter sequence required to trim a sequence = 2, maximum allowed error rate = 0.1. Also during this step, reads shorter than 36 bp were discarded.
Second, reads were mapped to the hg38 reference genome with Bowtie 2 v2.3.4.1.
Third, unmapped and low quality (MAPQ < 20) reads were excluded with samtools v1.8.
Forth, files were converted to bigWig format using bamCoverage v3.3.0 and signals visualized in UCSC genome browser after conversion to bigWig format. Bin sizes = 25 bases, Scaling/normalization method = 1X, Effective genome size = 2701495761, Extend reads to the given average fragment size = 150.
Genome_build: Human genome hg38 assembly
Supplementary_files_format_and_content: bigWig files were generated using bamCoverage v.3.3.0 function in Galaxy.
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| Submission date |
Mar 19, 2020 |
| Last update date |
Mar 03, 2023 |
| Contact name |
Claudio Gouveia Roque |
| E-mail(s) |
cag2230@cumc.columbia.edu
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| Organization name |
Columbia University
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| Department |
The Taub Institute for Research on Alzheimer's disease and the Aging Brain
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| Lab |
Ulrich Hengst
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| Street address |
650 West 168th Street
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE147205 |
Genome-wide DNA-binding program of CREB3L2-ATF4 heterodimers |
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| Relations |
| BioSample |
SAMN14403874 |
| SRA |
SRX7951452 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4420295_V5-CREB3L2-CREB3L2_Rep2.bigwig |
143.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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