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Sample GSM2082974 Query DataSets for GSM2082974
Status Public on May 15, 2016
Title Gli2 D0
Sample type SRA
 
Source name Gli2 chromatin binding at day 0 of M9-induced reprogramming
Organism Mus musculus
Characteristics cell type: tdMEFs at day 0 of reprogramming
chip antibody: Gli2 antibody (Abcam, 26056, lot GR223520-2)
Treatment protocol M9-induced neural reprogramming was performed in M9 medium (50% Neural basal, 50% DMEM/F12/Glutamax, 1xN2, 1xB27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, CHIR99021 3 mM, LDN193189 100 nM, A83-01 0.5 mM, Hh-Ag1.5 0.5 mM, retinoic acid 1 mM, SMER28 10 mM, RG108 10 mM, Parnate 2 mM, bFGF 10 ng/ml) at 5% O2 and 5% CO2 incubator at 37°C.
Growth protocol tdMEFs were cultured in MEF medium (Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax) in 5 % CO2 and 20 % O2 at 37 °C.
Extracted molecule genomic DNA
Extraction protocol Cells at indicated time points were cross-linked by 1% formaldehyde, quenched with 0.125 M glycine. The chromatin DNA was extracted and sheared into 200-400 bp, and immuoprecipitated using a EZ-ChIP kit (Millipore, 17-371), and indicated antibodies.
Barcoded ChIP-Seq libraries were made from 1 to 10 ng of chromatin immunoprecipitated DNA (ChIP-DNA) using the NuGen Ovation Ultralow V2 kit. 13 cycles were used for PCR amplification of the libraries. Amplified libraries were checked for adapter artifacts by Agilent Bioanalyzer using DNA high sensitivity reagents and chips (Agilent). All libraries were quantified by qPCR using an Illumina Library Quantification kit (KAPA Biosystems). Libraries were pooled in equimolar amounts and run on the HiSeq 2500 sequencer with a single read 50 cycle run (Illumina).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Data processing base-calling was done using CASAVA 1.8.4.
1. Reads are trimmed using the fastq-mcf program
2. The filtered reads are analyzed using the Fastqc program
3. The reads are aligned to the genome mm10 using bowtie2
4. Samtools is used to keep only the reads that do not map uniquely to the genome (mapq score >= 30)
5, bam2bed is used to convert bam files into bed files
6. Normalized Read intensity: The genome is divided into 20bp bins. The tag density is defined as the number of tags that map to within 75bp of each genomic bin. Tag density was then normalized according to method: ENCODE best practices; Landt et al., Genome Research 2012; (http://genome.cshlp.org/content/22/9/1813.full)
7. Peak Calling by comparing the normalized intensity with background tags (Paige et al., Cell 2012; (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462257/)). Enriched bins were merged.
Genome_build: mm10
Supplementary_files_format_and_content: Normalized intensity of the regions with at least one peak in any of the time points from Elk1 ChIP or Gli2 ChIP
 
Submission date Mar 07, 2016
Last update date May 15, 2019
Contact name Sheng Ding
Organization name THE J DAVID GLADSTONE INSTITUTES
Department Gladstone Institute of Cardiovascular Disease
Street address 1650 Owens Street
City SAN FRANCISCO
State/province California
ZIP/Postal code 94158
Country USA
 
Platform ID GPL17021
Series (2)
GSE78976 Genome-wide chromatin binding of Elk1 and Gli2 in chemical induced neural reprogramming
GSE78978 CHIP-seq analysis for chemical-induced neural stem cells from defined fibroblasts
Relations
BioSample SAMN04537315
SRA SRX1618619

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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