 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 19, 2017 |
| Title |
H3K27Ac ChIPseq in CTRL5_JL LCLs |
| Sample type |
SRA |
| |
|
| Source name |
CTRL5_JL_H3K27Ac
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Immortalized lymphoblastoid cell line (LC)
genotype/variation: control
chip antibody: H3K27Ac, Abcam ab4729
chip target: H3K27Ac
|
| Growth protocol |
LCL were cultured in RPMI 1640, FBS 15%, HEPES 1%, L-Glutamine 1%, Penicillin-Streptomycin 1%. All LCL samples were tested for mycoplasma.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as previously described (Frank et al., 2001) with some modification. Briefly, LCL were centrifuged at 150g for 5 minutes, an average of 150 x 106 LCLs were re-suspended in formaldehyde 1% in PBS for cross-linking. To stop the cross-linking reaction glycine was added to the final concentration 125 mM. Cells were re-suspended using ChIP SDS buffer (0.5% SDS, 5 mM EDTA, NaCl 100mM and 50 mM Tris-HCl at pH 8.1). Pellets were collected by centrifuging at 400g for 30 min and re-suspended in IP buffer (0.5% SDS, 5 mM EDTA, NaCl 100mM and 50 mM Tris-HCl at pH 8.6, Triton X-100 1,5%). To obtain a bulk of 300 bp DNA fragments, cells were sonicated using the Branson digital sonifier (Emerson Industrial Automation). Chromatin was quantified using Bradford protein assay (Bio-Rad). To immuno-precipitate YY1, 1 mg of total proteins was incubated with YY1 antibodies (sc-1703 or sc-281, Santa-Cruz Biotechnologies). For H3K27Ac chromatin immunoprecipitation, 100µg of proteins were immuno-precipitated using the antibody ab4729 (Abcam). Chromatin was incubated overnight with antibody at 4°C. G-Sepharose 4B (Thermo Fisher) beads were incubated with chromatin and antibodies mix for 4 hours at 4°C and followed by washes using low and high salt buffers (1% Triton X-100, 0.1% SDS, 150mM or 500mM NaCl , 2mM EDTA, 20 mM Tris-HCl ph8). Decross-linking was performed at 65°C for 3 hours. DNA was collected with QIAquick PCR Purification Kit (Qiagen).
Libraries were prepared as already described (Blecher-Gonen et al., 2013) with adaptations for the automated system Biomek FX.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
H3K27Ac ChIPseq in CTRL5_JL LCLs (unaffected control)
|
| Data processing |
Base calling and primary read filtering were performed according to Illumina protocols.
Reads were trimmed for potential adapter contamination using scythe 0.981 (min 4 nucleotides)
Trimmed reads were aligned to the GRCh38 genome using bowtie 1.0 (-v 2 -m 1).
Peaks were called using MACS 2.0.9 with default settings for YY1, and with the 'broad' option for H3K27ac and H3K27me3.
Genome_build: GRCh38
Supplementary_files_format_and_content: Peak calls (encodePeak format)
|
| |
|
| Submission date |
May 02, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Giuseppe Testa |
| E-mail(s) |
[email protected]
|
| Organization name |
European Institute of Oncology & University of Milano, Italy
|
| Lab |
Neurogenomics Research Centre
|
| Street address |
Via Adamello, 16
|
| City |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE98477 |
YY1 haploinsufficiency causes an intellectual disability syndrome featuring transcriptional and chromatin dysfunction [ChIP-seq] |
| GSE98478 |
YY1 haploinsufficiency causes an intellectual disability syndrome featuring transcriptional and chromatin dysfunction. |
|
| Relations |
| BioSample |
SAMN06883593 |
| SRA |
SRX2778368 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2597208_CTRL5_JL_IP_H3k27Ac_peaks.encodePeak.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
