 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 11, 2016 |
| Title |
MSC_H3K36me3Veh_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
marrow derived MSC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: marrow-derived mesenchymal stem cells
differentiation time point (day): 0
differentiation media: none
treatment: vehicle
antibody: H3K36me3 (Abcam, Ab9050, lot# GR10860-1)
|
| Treatment protocol |
Where indicated, MSC cells were treated with 100nM of 1,25(OH)2D3 for 3 hours prior to ChIP assay.
|
| Growth protocol |
Marrow derived MSC (mdMSC) were obtained from Dr. Janet Rubin, cryopreserved and expanded no more than 10 passages. The MSCs were cultured in minimum Eagle’s medium alpha (MEMα) modification supplemented with 10% fetal bovine serum from Hyclone (Logan, UT), and 1% penicillin-streptomycin from Invitrogen. For differentiation, cells were grown to confluency and then transferred to either osteogenic differentiation media (10 mM β–glycerophosphate and 50 µg/mL ascorbic acid) or adipogenic differentiation media (5 µg/mL insulin, 50 µM indomethacin, 0.1 µM dexamethasone) for the indicated lengths, replenishing the media every 2-3 days until assay.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as described previously (Meyer MB, Mol Endo. 2012). Briefly, samples were subjected to immuno-precipitation using either a control IgG antibody or the indicated experimental antibody (VDR, RXR, H3K4me1, H3K4me2, H3K4me3, H3K27Ac, H3K5Ac, H3K9Ac, H4K20me1, H3K36me3, or H3K9me3). To remove the calcified matrix from the differentiated cells, the cells were subjected to three 15 minute 300mM EDTA washes following fixation. Subsequently, matrix mix was subjected to 2x10 pulses with a Polytron Homogenizer (Power Gen 125, Fisher Scientific) while in NCP 1 buffer (Hepes 10mM pH 6.5, EDTA 10mM, EGTA 0.5mM, Triton X-100 0.25%). Resulting cell pellet was resuspended in NCP 2 buffer (Hepes 10mM pH 6.5, EDTA 1mM, EGTA 0.5mM, NaCl 200mM) and remainder of protocol was followed. The isolated DNA (or Input DNA acquired prior to precipitation) was then validated by quantitative real time PCR (qPCR) and further prepared for ChIP-seq analysis.
ChIP-seq libraries were prepared using the NEBNext DNA sample prep kit (NEB, #E6000L) with the Bioo NEXTflex ChIP-seq Barcodes (Bioo Scientific, Austin, TX, #514122) according to manufacturer’s protocols, with few exceptions. During the NEBNext prep, the Illumina adapters were replaced with the Bioo Scientific Barcoded adapters according to Bioo protocols. ChIP-DNA ligated libraries were cleaned up with Agencourt AMPureXP Magnetic Beads (Beckman-Coulter, #A63881). A pre-size selection PCR was performed using Phusion polymerase, NEXTflex Primer Mix and purified ligation product for 4 cycles of PCR according to the Bioo Protocol. Libraries were size selected using Invitrogen E-gels to a size of 400-500bp. Samples were then PCR amplified for 14 cycles using Phusion polymerase, NEXTflex Primer mix and the size selected DNA as per Bioo protocol, followed by Agencourt bead clean up. Libraries were validated for integrity using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Clusters were formed and sequenced on the Illumina HiSeq2000 sequencers by the University of Wisconsin-Madison DNA Sequencing Facility in the University of Wisconsin-Madison Biotechnology Center. DNA clusters were generated using a cBot Single Read Cluster Generation kit (ver. 3) on an Illumina cBot (Illumina, Carlsbad, CA) according to the manufacturer's instructions, to obtain an average of 1.5×108 clusters for each lane on a flowcell. All sequencing runs for 50mers were performed on an Illumina HiSeq2000 using the Illumina Sequencing kit (ver. 3). Fluorescent images were analyzed using the CASAVA 1.8.2 (Illumina Carlsbad, CA) to obtain FASTQ formatted sequence data. Twelve barcoded libraries were run per lane and this was repeated over 4 lanes. After which, the raw FASTQ for each sample was concatenated from the 4 lanes prior to mapping to create a single sample. The ChIP samples were repeated in biological replicate in the same manner (minimum of 2 replicates). Sequences were mapped to the mouse genome (mm9) using BOWTIE (--best –m 1) to yield unique alignments.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-seq runs were all 50bp. Barcoded samples were run over 4 lanes total and the fastq data were concatenated prior to BOWTIE mapping. 12 barcoded samples were run in each lane, over 4 lanes total. Barcodes were decoded by Illumina HiSeq2000 software automatically. All samples were mapped from fastq files using BOWTIE [-m 1 -- best] to mm9 [UCSCmouse genome build 9]. Replicate lanes were analyzed separately for reproducibility and normalization of the peak calls.
Peaks were called by using HOMER [http://biowhat.ucsd.edu/homer/] and MACS.
HOMER analysis was run using the default settings for peak finding. Histone peaks were called with a 2-fold enrichment over input instead of 4-fold (for transcription factors) given the nature of histone chip-seq.
False Discovery Rate (FDR) cut off was 0.001 (0.1%) for all peaks.
Genome_build: mm9
Supplementary_files_format_and_content: *.bed files contain the ChIP-seq peak locations, *.bedgraph files are for display in the UCSC genome browser
|
| |
|
| Submission date |
Apr 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark B Meyer |
| E-mail(s) |
markmeyer@wisc.edu
|
| Phone |
608-890-0857
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Nutritional Sciences
|
| Lab |
Meyer Lab
|
| Street address |
1415 Linden Dr.
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE79813 |
Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-Derived Mesenchymal Stem Cells (ChIP-seq) |
| GSE79815 |
Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-Derived Mesenchymal Stem Cells |
|
| Relations |
| BioSample |
SAMN04599599 |
| SRA |
SRX1672811 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2104147_MSC_H3K36me3Veh_rep2.peaks.bed.gz |
581.1 Kb |
(ftp)(http) |
BED |
| GSM2104147_MSC_H3K36me3Veh_rep2.ucsc.bedGraph.gz |
55.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
