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| Status |
Public on May 30, 2014 |
| Title |
H3K9me3_IDGSW3_d35 |
| Sample type |
SRA |
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| Source name |
H3K9me3_IDGSW3_d35
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| Organism |
Mus musculus |
| Characteristics |
cell line: IDG-SW3
cell type: osteocytic cells
differentiation day: Day 35
chip antibody: H3K9me3
chip antibody vendor: Abcam
chip antibody cat. #: Ab9989
chip antibody lot#: 777948
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| Treatment protocol |
For VDR and RXR ChIP-seq, IDG-SW3 cells were treated with 100nM of 1,25(OH)2D3 for 3 hours prior to ChIP assay.
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| Growth protocol |
Custom αMEM powdered tissue culture medium was purchased from Cellgro (Manassas, VA) and supplemented with 2.20g/L NaHCO3 (S233-3) (Fisher Scientific, Waltham, MA). Fetal Bovine Serum (FBS) (SH30396) was purchased from HyClone (Logan, Utah) and heat inactivated by incubation at 55°C for 30 minutes. Penicillin-Streptomycin Solution (SV30010) was purchased from Fisher Scientific, (Waltham, MA) and recombinant mouse interferon-gamma (rmIFN-γ) from Gibco/Invitrogen (Camarillo, CA). 10cm and 6 well collagen IV coated plates were purchased from BD Biosciences (San Jose, CA). IDG-SW3 cells (6) were expanded in 10cm collagen coated at 33°C in αMEM with 10% heat inactivated FBS, 100 units/mL penicillin/streptomycin, and 50U/mL IFNγ. Cells were harvested with 0.05% Trypsin/0.53mM EDTA (Fisher Scientific, Waltham, MA) and plated in 6 well collagen coated plates at 40,000 cells/cm2. To induce osteocytogenesis, two days after plating, cells were transferred to 37°C and media was changed to osteogenic media (αMEM with 10% heat inactivated FBS, 100 units/mL penicillin/streptomycin, 50μg/mL ascorbic acid (A4034, Sigma, St. Louis, MO) and 4mM β-glycerol phosphate (G9422, Sigma, St. Louis, MO) without IFNγ. Osteogenic media was changed three times weekly.
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| 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 transcription factor ChIP sample was repeated in biological replicate in the same manner (minimum of 2 replicates). Histone modifications were analyzed as single samples. Sequences were mapped to the mouse genome (mm9) using BOWTIE (--best –m 1) to yield unique alignments.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| 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 QuEST [http://mendel.stanford.edu/sidowlab/downloads/quest/].
QuEST 2.4 was run using the recommend settings for transcription factor (TF) like binding with the following exceptions: kde_bandwith=30, region_size=600, ChIP threshold=30, enrichment fold=3, rescue fold=3
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
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| Submission date |
Feb 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark B Meyer |
| E-mail(s) |
markmeyer@wisc.edu
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| Phone |
608-890-0857
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| Organization name |
University of Wisconsin-Madison
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| Department |
Nutritional Sciences
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| Lab |
Meyer Lab
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| Street address |
1415 Linden Dr.
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE54782 |
The Osteoblast to Osteocyte Transition: Epigenetic Changes and Response to the Vitamin D3 Hormone [ChIP-seq] |
| GSE54784 |
The Osteoblast to Osteocyte Transition: Epigenetic Changes and Response to the Vitamin D3 Hormone |
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| Relations |
| BioSample |
SAMN02630834 |
| SRA |
SRX467385 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1323940_IDGSW3D35_H3K9me3Veh_rep1_nb_BIOO.peaks.bed.gz |
606.7 Kb |
(ftp)(http) |
BED |
| GSM1323940_IDGSW3D35_H3K9me3Veh_rep1_nb_BIOO.tagdirnew.ucsc.bedGraph.gz |
65.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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