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| Status |
Public on Nov 19, 2015 |
| Title |
TAF15 |
| Sample type |
SRA |
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| Source name |
HEK293 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
antibody: TAF15
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| Treatment protocol |
The protocol used was merged from [75], [54] and Magnify chromatin immunoprecipitation system (Invitrogen). Briefly, HEK-293 cells (1x107per 10 cm petri dish) were cross-linked directly on plates by adding 37% formaldehyde to the media in a 1.4% final concentration for 15 min at room temperature. Cross-linking was stopped by adding glycine (final concentration 125 mM) followed by incubation for 5 min, at room temperature. Cells were scraped off in 500 µl of ice-cold 2x Phosphate Buffered Saline (PBS) and transferred to eppendorf-tubes and placed on ice. Cells were centrifuged at 2,000 g for 5 min, at 4°C, and washed twice with ice-cold PBS. Cells were lysed in 500 µl of ice-cold immunoprecipitation (IP)-Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1% Triton X-100, 0.5% NP40) supplemented with Complete Mini protease inhibitor cocktail (Roche), hereafter referred to as supplemented IP-Buffer. Samples were centrifuged at 12,000 g for 1 min, at 4°C, and washed once in ice-cold supplemented IP-Buffer. 500 µl of ice-cold supplemented IP-Buffer were added to the pelleted nuclei and sample re-suspended. Chromatin was fragmented by sonication using a Bioruptor (Diagnode) at high effect (80 cycles, 30s on plus 30s off) to generate fragments of 150-300 bp. Fragment lengths were examined by agarose gel electrophoresis. Samples were centrifuged at 12,000 g for 10 min, at 4°C. Supernatants were transferred to fresh ice-cold tubes and stored at -80°C.
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| Growth protocol |
HEK-293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum, streptomycin (2.0 g/l), penicillin (1.2 g/l) and glutamine (0.3 g/l) (complete DMEM). HEK-293 cells were obtained from the American Type Culture Collection (CRL-1573). Cells were grown at 37 °C and 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP experiment, material corresponding to 5x106 starting cells was used. Samples were diluted with an equal volume of D-Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1% Triton X-100). 5 µg of antibody (FUS sc-47711 Santa Cruz and EWS sc-48404 Santa Cruz) or 2 µg antibody for acetylated histone protein (ac-H3K9 ab4441-50 Abcam) were used for each ChIP. Samples were incubated in an ultrasonic water bath for 25 min, at 4°C, and afterwards centrifuged at 12,000 g for 10 min, at 4°C. 20 µl magnetic AG-beads (Invitrogen) were used for each ChIP. Beads were washed 3 times in 1 ml of supplemented IP-Buffer before use. After the final wash, beads were suspended in 40 µl of supplemented IP-Buffer and mixed with 90% of the chromatin supernatant described above. The mix was incubated in rotation overnight, at 4°C. Beads were washed twice with 200 µl of ice-cold supplemented IP-Buffer and three times with 200 µl ice-cold D-Buffer supplemented with protease inhibitors. Cross-linking was reversed by adding Reverse X-link Buffer and Protease K (Invitrogen) and incubating in water bath for 20 min, at 55°C. The remaining 10% of the reversed cross-linking chromatin supernatant was used as input. Supernatants were transferred to fresh tubes and incubated at 95°C to inactivate Protease K. DNA was purified using DNA Purification Buffer plus DNA purification magnetic beads, as recommended (Invitrogen). DNA was stored at -20°C.
DNA from FUS, EWS, and Ac-H3K9 ChIP experiments, as well as the corresponding input, was sequenced using the Illumina Hiseq 2000 platform at BGI in Shenzhen, China. Library preparation, cluster generation and sequencing by synthesis were performed according to manufacturer's protocol. All raw reads were aligned using Burrows-Wheeler Aligner (BWA version 0.5.8c (r1536) [48]) to the human reference genome (hg19). Aligned reads were processed by Model-based Analysis of ChIP-Seq (MACS) 1.4.0rc2 [49] for peak calling. Significant peaks were defined using the criteria of a threshold of minimally 9 reads and p-value less than 10-8 as suggested previously [50]. Input ChIP DNA was used as negative control.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
ChIP-seq reads were aligned to the human genome hg19 assembly using BWA (0.6.1-r104) with default "aln" options.
Find peaks using MACS (version 14) with the following options: -g hs -w --call-subpeaks
Genome_build: hg19
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| Submission date |
Sep 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Yonglun Luo |
| E-mail(s) |
alun@biomed.au.dk
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| Phone |
0045-22411944
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| Organization name |
Aarhus University
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| Street address |
Wilhelm Meyers Allé 4
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| City |
aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE73492 |
EWS and FUS Bind a Subset of Transcribed Genes Encoding Proteins Enriched in RNA Regulatory Functions |
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| Relations |
| SRA |
SRX1293072 |
| BioSample |
SAMN04121939 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1895987_JB3_JB6_MACS_wiggle.tar.gz |
429.7 Mb |
(ftp)(http) |
TAR |
| GSM1895987_JB3_JB6_peaks.bed.gz |
3.9 Kb |
(ftp)(http) |
BED |
| GSM1895987_JB3_JB6_peaks.xls.gz |
6.1 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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