|
| Status |
Public on May 13, 2016 |
| Title |
Input_DNA_H3.3K36M |
| Sample type |
SRA |
| |
|
| Source name |
C3H10T1/2 cells & S2 cells
|
| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell type: C3H10T1/2: C3H embryo-derived mesenchymal progenitor cells; S2: derived from a primary culture of late stage (20-24 hours old) Drosophila melanogaster embryos
genotype: C3H10T1/2: Stably expressing FLAG-HA-tagged K36M mutant H3.1; S2: None
passage: C3H10T1/2: Passage 10-15; S2: unknown
|
| Growth protocol |
C3H10T1/2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, CellGro). Drosophila S2 cells were cultured in Schneiderís Drosophila Medium (Invitrogen) containing 10% heat-inactivated FBS (CellGro).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
~2x10^7 C3H10T1/2 cells were lysed using digesting buffer (50 mM Tris-HCl pH 7.6, 1 mM CaCl2 and 0.2% Triton X-100) and digested with micrococcal nuclease to obtain mono-nucleosomes. The chromatin were then dialyzed into RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100) for 2 h at 4°C. After centrifugation, soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5% of the mouse cell chromatin. The mixed soluble chromatin was incubated with αH3K36me3 (Active Motif, 61101), αH3K36me2 (Millipore, 07-369) or αH3K27me3 (Cell Signaling Tech, 9733) antibody bound to 75 μl protein A or protein G Dynal magnetic beads (Invitrogen) and incubated overnight at 4°C, with 5% kept as input DNA. Magnetic beads were washed with RIPA buffer and LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate) and chromatin was eluted. ChIP DNA was treated with Proteinase K (Roche) and recovered using Qiagen PCR purification kit.
libraries were prepared according to the Illumina TruSeq protocol
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
5% input DNA
mouse C3H10T1/2 cells and Drosophila S2 cells were mixed in 1:1 ratio prior to library preparation
|
| Data processing |
a custom genome, termed mm9-dm6, was created by combining both Mus musculus genome built, mm9 and Drosophila melanogaster genome built, dm6
Reads that passed the quality score were aligned to mm9_dm6 custom genome using bowtie 1.0.0 with default parameters
Sample normalization factors (Rx) for reads aligned to mouse genome were determined as 1 over the number of reads mapping to Drosophila genome per million base-pair
Genome_build: mm9-dm6 custom genome
Supplementary_files_format_and_content: tab-separated abundance measurement containing the reference (reads aligned to dm6 genome) normalized intensities in each sample
|
| |
|
| Submission date |
Dec 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter W Lewis |
| E-mail(s) |
pwlewis2@wisc.edu
|
| Phone |
608-316-4388
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Department of Biomolecular Chemistry
|
| Lab |
Room 2174, Wisconsin Institute for Discovery
|
| Street address |
330 N Orchard Street
|
| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53715 |
| Country |
USA |
| |
|
| Platform ID |
GPL21227 |
| Series (2) |
| GSE69291 |
H3K36 mutations promote sarcomagenesis through genome-wide remodeling of H3K36 and H3K27 methylation |
| GSE75855 |
H3K36 mutations promote sarcomagenesis through genome-wide remodeling of H3K36 and H3K27 methylation [H31_ChIP-Rx] |
|
| Relations |
| BioSample |
SAMN04328025 |
| SRA |
SRX1473021 |
