 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 01, 2019 |
| Title |
siYAP_TAZ_H3K27ac_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
HEK293
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
developmental stage: embryonic
tissue: kidney
chip antibody: Rabbit polyclonal anti-H3K27ac Active Motif Cat#39133 Lot#31814008
sirna transfection: Silencer Select siRNA YAP1, ThermoFisher Assay ID: s534572; Silencer Select siRNA WWTR1 ThermoFisher Assay ID: s24788
|
| Treatment protocol |
siRNA transfections were performed using Invitrogen RNAiMAX reverse transfection protocol. Cells were plated in antibiotic free 10% FBS DMEM containing indicated Ambion/ThermoFisher Silencer Select siRNA for a final concentration of 10nM that was pre-incubated in lipofectamine RNAiMAX reagent in Opti-MEM.
|
| Growth protocol |
HEK293 (human embryonic kidney cell line) were maintained at 37°C in a 5% CO2 incubator in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq was performed using 1x107 siRNA transfected HEK293. Cells were transfected for 1 or 4 days for H3K18ac (814) and H3K27ac (Active Motif) ChIP, 4 days for H3K4me1 (abcam), RAD21 (abcam), and 8 days for YAP (CST D8H1X), TEAD1 (BD Transduction Laboratories), and TEAD4 (Santa Cruz N-G2). For H3K18ac, H3K27ac, H3K4me1 and RAD21 ChIP-seqs cells were cross-linked for 1% formaldehyde for 10 minutes at room temperature on rotator. Formaldehyde crosslinking was quenched with 0.14M glycine for 30 minutes at room temperature on rotator. Cells were washed with PBS and scraped from plates in PBS with Roche protease inhibitor cocktail. Cells were pelleted and lysed in 400uL lysis buffer (1% SDS, 50mM Tris-HCl pH8, 20mM EDTA, Roche complete protease inhibitors) and sonicated at 4°C using the Qsonica Q800R2 at 20% amplitude 10s on 30s off until DNA fragments from sheared chromatin were mostly between the sizes of mostly ~200-600 base pairs. Samples were normalized for equal amounts of DNA as measured by Qubit fluorometer in sonicated, cross-linked chromatin prior to pre-clear and IP. Up to 100uL of sonicated chromatin was diluted in 10X lysis dilution buffer (16.7 mM Tris-HCl, 1.1% Triton X-100, 1.2mM EDTA, 167mM NaCl) and precleared for 1h 4°C with 30uL of protein A dynabeads washed 10X lysis dilution buffer on nutator. IPs were performed O/N at 4°C on nutator with precleared chromatin and 2ug of antibody or 5uL of H3K18ac anti-rabbit sera. 50uL of protein A dynabeads were added for 4h on nutator at 4°C. Bead-immunocomplexes were washed for 5min 2X with each of the following buffers in order: wash buffer A (50mM Hepes pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), wash buffer B (50mM Hepes pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 500mM NaCl), LiCl buffer (20mM Tris-HCl pH8, 0.5% NP-40, 0.5% Deoxycholate, 1mM EDTA, 250mM LiCl), TE (50mM Tris-HCl pH8, 1mM EDTA). Elution was performed in 150uL of elution buffer (50mM Tris HCl pH8, 1mM EDTA, 1% SDS) then ChIP samples and inputs (10uL of precleared chromatin lysis plus 140uL elution buffer) were reverse crosslinked O/N at 65°C. Samples were RNase A treated for 1h at 37°C and DNA was purified and extracted with phenol/chloroform and ethanol precipitated. DNA pellets were resuspended in 12uL of TE and measured using Qubit fluorometer. YAP, TEAD1, and TEAD4 ChIP-seqs were performed similarly with the following modifications: cells were double-crosslinked with 4mM DSG in PBS for 30min then 1% formaldehyde for 10 min, crosslinking was quenched in 500mM Tris pH7.9 for 20min and cell pellets were lysed in 1mL lysis buffer 1 (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, Roche cOmplete protease inhibitors) for 10min on ice. Lysate was pelleted at 3000 rpm 5min 4°C then resuspended in 1mL lysis buffer 2 (10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, Roche complete protease inhibitors) and placed on nutator 10min 4°C and pelleted as before, then resuspended in 125uL of lysis buffer 3 (10mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, Roche complete protease inhibitors) and sonicated, 2ug of antibody was used, magnetic beads were washed and blocked in 0.5% BSA in PBS.
ChIP sequencing libraries were constructed from 1 ng of immunoprecipitated and input DNA using the KAPA Hyper Prep Kit from KAPA Biosystems and NEXTflex ChIP-Seq barcodes purchased from Bioo Scientific.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Reads were mapped to the hg19 human genome reference using Bowtie2 software. Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were retained for further analysis. Duplicate reads that mapped to the same exact location in the genome were counted only once to reduce clonal amplification effects.
A custom algorithm executed by MATLAB was used for further processing. The genome was tiled into 50 base pair windows and each read was extended by 150 bases and was counted as one read to each window to which it partially or fully matched. The total counts of the input and ChIP samples were normalized to each other.
Wiggle files were generated using a custom algorithm and present the data as normalized tag density.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Apr 22, 2019 |
| Last update date |
Jun 02, 2019 |
| Contact name |
Bing Ren |
| E-mail(s) |
biren@health.ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE130135 |
De-differentiation by Adenovirus E1A Due to Inactivation of Hippo Pathway Effectors YAP and TAZ [ChIP-seq] |
| GSE130137 |
De-differentiation by Adenovirus E1A Due to Inactivation of Hippo Pathway Effectors YAP and TAZ |
|
| Relations |
| BioSample |
SAMN11476386 |
| SRA |
SRX5718161 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3732660_siYAP_TAZ_H3K27ac_ChIPseq.bigwig |
153.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
