|
| Status |
Public on Nov 10, 2015 |
| Title |
Input control for PDX1 H3K4Me2 and H3K27Ac ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
input control
|
| Organism |
Mus musculus |
| Characteristics |
cell line: BetaTC6
|
| Biomaterial provider |
ATCC Cat #CRL-11506
|
| Treatment protocol |
Cells were harvested when they reached confluence.
|
| Growth protocol |
Beta-TC6 cells were purchased from ATCC (CRL-11506) and cultured in DMEM supplemented with 15% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. All cells used in experiments were at fewer than 15 passages.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Beta-TC6 cells (~40-160 million) were fixed for 30 minutes in 2mM DSG and for 10 minutes in 1% formaldehyde and then either frozen at -80ûC or processed immediately. Nuclei were isolated in buffer containing 1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.0, and protease inhibitors and sonicated using a Diagenode Bioruptor to shear chromatin to 200-1000bp fragments. Protein-DNA complexes were incubated with antibodies against BMAL1 and CLOCK (affinity-purified guinea pig IgGs as described above), H3K4Me2 (Abcam), H3K27Ac (Active Motif), H2AZ (Active Motif), or PDX1 (Novus Biologicals) and immunoprecipitated with IgG paramagnetic beads (Invitrogen). Eluted chromatin was isolated using MinElute PCR purification columns (Qiagen).
Sequencing libraries were generated using KAPA DNA Library Preparation kits (Kapa Biosystems, KK8504) according to manufacturer's instructions. Library concentrations were assessed by both a Bioanalyzer (Agilent) and qPCR-based quantification (Kapa Biosystems)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Antibody: none (input)
|
| Data processing |
Alignment: Raw sequence reads were aligned to the mm10 reference genome and displayed using UCSC annotated genes using bowtie version 1.1.1 with parameters “--best” and “-m 1” to ensure reporting of uniquely mapped reads (tags)
Peak calling: Peaks were called using Homer “findPeaks” and specifying “-style factor” for BMAL1, CLOCK, and PDX1 and “-style histone” for H2A.Z, H3K4Me2, and H3K27A. ChIP-Seq peaks were designated as regions with 4-fold enrichment over both the input sample and the local background and were normalized to 10 million reads using default parameters.
Genome_build: mm10
Supplementary_files_format_and_content: Peaks that were identified with Homer; in BED format
|
| |
|
| Submission date |
Jul 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark Perelis |
| E-mail(s) |
mperelis@u.northwestern.edu
|
| Organization name |
Northwestern University
|
| Street address |
303 E Superior Street Lurie Research Center Rm 7-220
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE70960 |
Pancreatic Beta-Cell Enhancers Regulate Rhythmic Transcription of Exocyst Triggering and Diabetes [ChIP-seq] |
| GSE70961 |
Pancreatic Beta Cell Enhancers Regulate Rhythmic Transcription of Exocyst Triggering and Diabetes |
|
| Relations |
| BioSample |
SAMN03861976 |
| SRA |
SRX1098146 |
