 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 05, 2014 |
| Title |
Standart H3K4me2 ChIP-Seq (2*1.00E+06 cells) replica7 |
| Sample type |
SRA |
| |
|
| Source name |
mouse derived lymphoblastic cell line (D10.G4.1 cells, ATCC)
|
| Organism |
Mus musculus |
| Characteristics |
strain: AKR/J
cell type: D10.G4.1 cells, ATCC
chip antibody: H3K4me2 (clone Y47, lot#YH041501C; Abcam)
cell count: 2 million cells
|
| Growth protocol |
The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: 10% T-STIM with Con A (rat IL-2 culture supplement ) Becton Dickinson, Catalog No. 354115), 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 10 pg/ml mouse IL-1 alpha (R&D Systems, Catalog No. 400ML). Subculture actively growing suspension cultures before they have reached 1 X 106 cells/mL or the IL-1 and IL-2 will be rapidly depleted and the cells will lose viability. Use inoculation densities of 2 X 105 viable cells/mL. Medium Renewal:Twice per week.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed and fixed for 10 min at room temperature with 1:10 dilution of 11% formaldehyde buffer, neutralized 2.5 M glycine, washed twice in ice cold PBS, and cell pellets snap frozen in liquid nitrogen before storing at -80°C. Frozen cells were lysed in 120 μl of SDS lysis buffer containing 1 mM PMSF, 20 mg/ml sodium butyrate and proteinase inhibitor cocktail, and chromatin sheared by sonication, using Bioruptor. Different amount of chromatin was used for immuno-precipitation. Chromatin was diluted in 1ml of radioimmunoprecipitation assay buffer and immunoprecipitation was done overnight at 4°C by incubating 1 μl of antibody pre-coated on 10 μl protein A coated magnetic beads. Immunocomplexes were captured and washed with RIPA buffer x1, high-salt buffer x1, LiCl buffer x1, and low salt buffer x1. Beads were resuspended in TE transferred to fresh tubes, captured and then resuspended in 1% SDS elution buffer. The crosslinking was reversed by incubating samples for 15 minutes at 65°C, and treated with 2 μl of RNase A for 30 minutes at 37°C, and, with proteinase K for 6 hours at 55°C followed by overnight incubation at 65°C. DNA was purified using affinity. The amount of DNA in the ChIP samples was quantified using picogreen assay. One ng of ChIP DNA was PCR amplified for 18 to 22 cycles using whole genome amplification (WGA). Two μg of this amplified DNA was treated with restriction enzyme to remove the WGA primer sequences and then purified using AmpureXP bead. DNA was sonicated with E220 Covaris multiplex sonicator (Covaris) to generate 100-250bp DNA fragments.
Libraries were prepared according to SOLiD's instructions accompanying the DNA Sample Kit (5500 SOLiD® Fragment 48 Library Core Kit & Fragment Library Barcode Adaptors 1-96).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500 Genetic Analyzer |
| |
|
| Description |
Standart_H3K4me2_2mln_cells_rep7
|
| Data processing |
ChIP-seq data (csfasta and qual files) were mapped against the mouse reference genome mm10 (downloaded from the Genome Bioinformatics database of the University of California Santa Cruz) using Bowtie v.0.12.7.
Pairwise Spearman correlation and scatterplots between samples were calculated by comparing the sequencing coverage at genome wide 500bp windows using MEDIPS v.1.10.0 (extend = 120, uniq = F, window_size=500, BSgenome ="BSgenome.Mmusculus.UCSC.mm10")
For the correlation analysis of Standart H3K4me2 ChiP-Seq replicas 1-6, we have trimmed reads from 50bp to 35bp. Row files contain untimmed reads.
Genome_build: mm10
Supplementary_files_format_and_content: For visualization of the ChIP-seq data in public genome browsers, sequencing coverage was calculated at genome wide 50bp windows after extending each read to a length of 120bp along the sequencing direction using MEDIPS v.1.10.0 (extend = 120, uniq = F, window_size=50, BSgenome = "BSgenome.Mmusculus.UCSC.mm10") and the resulting coverage profiles (RPKM) were exported as wiggle files. Conversion from wiggle to bigwig files using wigToBigWig utility provided by UCSC Genome Browser
|
| |
|
| Submission date |
Dec 24, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Anna Gerasimova |
| Organization name |
La Jolla Institute for Allergy and Immunology
|
| Street address |
9420 Athena Circle
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL16790 |
| Series (2) |
| GSE53644 |
Reproducibility of standard and micro-scaled H3K4me2 ChIP-Seq assay in mouse derived lymphoblastic cell line (D10.G4.1 cells, ATCC) |
| GSE53646 |
Epigenomic analysis of primary human T cells reveals novel enhancers associated with Th2 memory differentiation and asthma susceptibility |
|
| Relations |
| BioSample |
SAMN02486147 |
| SRA |
SRX399015 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1298060_Standart_H3K4me2_2mln_cells_rep7.bw |
54.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
