|
| Status |
Public on Jul 23, 2015 |
| Title |
IgG_MyoD-2_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
Primary proliferating myoblasts
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: muscle
cell type: primary
days in culture: 3
chip antibody: mouse IgG (278-010, Ancell)
|
| Treatment protocol |
Cell were fixed with formaldehyde and nuclear lysate were made
|
| Growth protocol |
We isolated SC from gastrocnemius muscles of 2-3 weeks old C57bl/6 WT mice. Cells were grown at 37°C, 5% CO2 on GHR MatrigelTM (BD Biosciences, USA) in proliferation medium (BIOAMF-2, Biological Industries, Israel), which was replaced daily. For MyoG ChIP-seq, PMs were differntiate for 24hr: cells were grown as above to 75-80% confluency and then induced to differentiate by serum starvation in differentiation media (DM): DMEM containing 4% horse serum (Gibco, UK) and 0.04U/mL human Insulin. Each transcription factor experiment was performed in biological replicates.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei (sonicated to yield DNA fragments of ~300bp) and TF-DNA complexes were isolated with relevant antibody, washed and DNA was purified using Qiagen Minelute kit.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
second IP experiement of mouse IgG (for MyoD)
|
| Data processing |
For Samples 1-4 and 13-16, basecalls were performed using CASAVA version 1.8.2. For Samples 5-12, basecalls were performed using CASAVA version 1.7.0.
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie with the following options: -a -m 1 --best --strata -v 1.
Peaks of enriched binding regions were detected by merging biological replicates of IP and NIS or IgG. Peaks were called using MACS1.4.
Genome_build: mm9
Supplementary_files_format_and_content: Bed files containing peaks detected by MACS1.4.
|
| |
|
| Submission date |
Mar 20, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
kfir baruch Umansky |
| E-mail(s) |
kfirum@weizmann.ac.il
|
| Phone |
97289342318
|
| Organization name |
Weizmann Institute
|
| Street address |
POB 25
|
| City |
Rehovot |
| State/province |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE56077 |
Transcription factor Runx1 cooperates with MyoD and c-Jun to regulate the balance of myoblast proliferation/differentiation [ChIP-seq] |
| GSE56131 |
Transcription factor Runx1 cooperates with MyoD and c-Jun to regulate the balance of myoblast proliferation/differentiation |
|
| Relations |
| BioSample |
SAMN02693128 |
| SRA |
SRX497485 |
