 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 26, 2018 |
| Title |
H3K36me2 ChIP-seq in G34W |
| Sample type |
SRA |
| |
|
| Source name |
HeLa_G34W H3.3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
genotype/variation: stably expressing mutant (G34W) H3.3
chip antibody: H3K36me2
chip antibody vendor/cat.#: Abcam, Ab9049
|
| Treatment protocol |
For infections, Hela cells were incubated with viral supernatants in the presence of 8 µg/ml polybrene for 14 hr. After 48 hr, the infected cells were selected with blasticidin (5 µg/ml) for overexpression experiments.
|
| Growth protocol |
Hela cells were maintained in DMEM media [Dulbecco’s modified Eagle’s medium (DMEM, Cellgro) supplemented with 10% Fetal Bovine Serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, 292 µg/ml L-glutamine]. Media was changed every other day.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq were performed following the protocol: Using ChIP-seq technology to generate high-resolution profiles of histone modifications. O'Geen H, Echipare L, Farnham PJ. Methods Mol Biol. 2011;791:265-86. doi: 10.1007/978-1-61779-316-5_20.
ChIP analysis was performed as described previously (Li et al., 2014) with minor modification. In brief, cells were cross-linked with 1% formaldehyde for 10 min and stopped with 125 mM glycine for 5 min. Fixed cells were scraped from dishes and washed with PBS for 2 times. Cell pellets were resuspended with cell lysis buffer (5 mM PIEPES pH 8.0, 85 mM KCl, 1% NP-40) with protease inhibitor cocktail (Roche) and 10 mM phenylmethanesulfonyl fluoride (PMSF). After 20 min rotation in 4 °C, cell nuclei were pelleted by centrifugation. The nuclei were resuspended with nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) containing protease inhibitors and sonicated using Bioruptor Sonicator (Diagenode). Length of most sheared DNA fragments were between 100-400 base pairs. Sonicated chromatin contains approximately 20 μg of DNA were diluted 4 fold with dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.01% SDS) and were subjected to immunoprecipitation with 2 μg of antibodies overnight at 4 °C. Protein A/G beads (Millipore) were then added and incubated for 1 hr. The immunoprecipitates were washed twice with low-salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS) and high-salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS) and then once with LiCl buffer (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40, 1% Na-Deoxycholate). DNA was eluted with vigorously shaking in elution buffer (50 mM NaHCO3, 1% SDS) for 15 min and then reverse crosslink in the presence of 0.3M NaCl at 67 °C for 12 hr. DNA was purified using PCR purification kit (Qiagen) and analyzed by quantitative real-time PCR on the ABI 7500-FAST System using the Power SYBR Green PCR Master Mix (Applied Biosystems).
For ChIP-seq, sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer's protocol. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor) were band isolated from an agarose gel. DNA fragments were sequenced using single-end sequencing technology on Illumina HiSeq 3000 platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
Hela_W_36me2
|
| Data processing |
Basecalls performed by Illumina CASAVA 1.8.2
ChIP-seq reads were aligned to the hg19 genome using Bowtie v1.1.0
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq wig files were generated using MACS-1.4.2; Scores represent the ChIP-seq tag numbers
|
| |
|
| Submission date |
Feb 08, 2018 |
| Last update date |
Apr 26, 2018 |
| Contact name |
Jiejun Shi |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California Irvine
|
| Department |
School of Medicine
|
| Lab |
Li lab
|
| Street address |
5270 California Ave
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92617 |
| Country |
USA |
| |
|
| Platform ID |
GPL21290 |
| Series (1) |
| GSE110389 |
Histone H3.3 G34 mutations alter histone H3K36 and H3K27 methylation in cis |
|
| Relations |
| BioSample |
SAMN08511647 |
| SRA |
SRX3675861 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2990421_Hela_W_36me2.bw |
406.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
