 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 14, 2022 |
| Title |
HepaRG d30DMSO H3K4me3 Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HepaRG cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: differentiated HepaRG cells day 30
antibody: H3K4me3
agent: DMSO
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Detached HepaRG cells were fixed with 1% formaldehyde in William’s E medium for 10 min. Crosslinking was stopped by adding glycine to a final concentration of 125 mM for 5 min at room temperature. If not mentioned otherwise samples were thereafter kept on ice or at 4 °C and centrifugations were performed at 800 x g for 5 min. After washing twice with 125 mM glycine in PBS and once in PBS, cells were resuspended consecutively in 5 ml buffer A (10 mM HEPES-NaOH, 10 mM EDTA, 0.5 mM EGTA, 0.25 % Triton X-100, 1 x PIC (Complete Protease Inhibitor Cocktail Tablets)), respectively buffer B (10 mM HEPES-NaOH, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01 % Triton X-100, 1 x PIC) and rotated for 10 min followed by centrifugation each. Cells were resuspended in 600 μl buffer C (25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS, 1 x PIC). Sonication of chromatin into fragments between 100 and 800 bp was performed using the Bioruptor NGS (Diagenode, Liège, Belgium) on high power setting in 200 μl volumes, either in 0.5 ml shearing tubes with 30 cycles or in 1.5 ml microtubes (Diagenode, Liège, Belgium) with 10 cycles and each cycle consisting of 30 seconds sonication and 30 seconds recovery time. Samples were diluted with double volume dilution buffer (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.75 % Glycerol).
For each immunoprecipitation 10 µl protein G beads (Invitrogen, Carlsbad, USA) were combined either with 2 μg H3K4me3 (Diagenode, Liège, Belgium, pAb-003-050), H3K27ac (pAb-196-050) or H3K4me1 (pAb-194-050) antibody, 5 μl BSA (NEB, Frankfurt am Main, Germany, 10 mg/ml) and rotated for 2 h. Then, 300 μl sonicated chromatin (equivalent to 1 million cells) were added. Immunoprecipitations of H3K27ac were supplemented with Na-Butyrate to a final concentration of 10 mM. After rotation overnight beads were consecutively washed with 1 ml of WB1 (20 mM Tris-HCl, (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), twice WB2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), WB3 (10 mM Tris-HCl, (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5 % Nonident P-40, 0.5 % Sodiumdesoxycholate) and twice TE / NaCl (10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA) under rotation for 10 min. Elution was performed using 200 µl EB (100 mM NaHCO3, 1 % SDS) for 30 min in a shaking device at room temperature. Eluates were supplemented with crosslink reversal solution (4 μl 5 M NaCl, 2 μl 0.5 M EDTA and 2.5 μl Proteinase K (20 mg/ml)) and incubated overnight at 55 °C. ChIP DNA was purified by phenol chloroform extraction followed by ethanol precipitation. Library preparation was performed using the Truseq ChIP Sample Preparation Kit (Illumina, San Diego, USA) following the manual except for omitting size selection. All ChIP-seq libraries were sequenced 2 x 100 bp paired-end on a HiSeq2500 (Illumina, San Diego, USA).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Low quality ends (phred score=20) of FastQ format reads were trimmed in addition to adapter removal using Trim Galore (version 0.3.3) [http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/]. Trimmed reads were aligned to the human reference genome (hs37d5) using GEM mapper (version 1.376 beta) (Marco-Sola et al., 2012). Samtools (version 1.3) was used to convert SAM to BAM format (H. Li et al., 2009). MarkDuplicate (version 1.115) from Picard tools [http://broadinstitute.github.io/picard/] was used to mark PCR duplications. Differential analysis and annotation of differential sites (DERs) to associated genes and promoter regions were performed using diffReps (version 1.55.5) with padj<0.001 and |FC|>2 (Shen et al., 2013). A circle plot of differential sites was generated using Circlize (version 0.3.10) (Gu et al., 2014). GREAT (version 3.0.0) was used for prediction of associated GO terms with default extension settings (binominal p-value<0.05) (McLean et al., 2010). ChIP-seq coverage tracks normalized to 1 X sequencing depth and mean coverage plots around TSS (bin size = 50 bp) were generated with deepTools (Ramírez et al., 2014).
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files containing ChIP-seq signals normalized to sequencing depth
|
| |
|
| Submission date |
Jul 13, 2021 |
| Last update date |
Jul 14, 2022 |
| Contact name |
Kathrin Kattler-Lackes |
| E-mail(s) |
Kathrin.Kattler@uni-saarland.de
|
| Phone |
06813022443
|
| Organization name |
Saarland University
|
| Department |
Epigenetics
|
| Street address |
Campus A2.4 Raum 0.13
|
| City |
Saarbrücken |
| State/province |
non-US Resident |
| ZIP/Postal code |
66123 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE179988 |
Epigenetic consequences of DMSO treatment on the HepaRG cell line |
|
| Relations |
| BioSample |
SAMN20189105 |
| SRA |
SRX11424563 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5444552_HepaRG_d30+_H3K4me3_R1.coverage.bw |
57.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
