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Sample GSM1842754

Query DataSets for GSM1842754

Status

Public on Aug 05, 2015

Title

mES_Nanog_3p_2i_r2

Sample type

SRA

Source name

KH2 mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell type: mouse embryonic stem cells
culture condition: 2i for 3 passages
antibody epiptope: Nanog
antibody-vendor: Cosmo Bio Ltd
antibody-catalog-nr: REC-RCAB0002P-F
antibody-lot: 20100713

Growth protocol

Mouse embryonic stem cells (mESCs) were cultured in regular media containing 15% FBS, 1% PEN/STREP, 1% Glutamine, 1% NEAA and LIF. For ES cell maintenance dishes were coated with 0.2% gelantin and irradiated CF1 mouse embryonic fibroblasts (MEFs) were plated as confluent layer of feeder cells. ESCs were seeded in a density of 50000 cells/ 6well and were splitted every 2-3 days. The 2i media was prepared according to the following protocol (Ying et al., 2008) and was supplemented with LIF.

Extracted molecule

genomic DNA

Extraction protocol

RNA-Seq: Polyadenylated RNA was isolated using Olig dT beads (Invitrogen). RNA was fragmented to a size of 200-600 base pairs, followed by ligation to RNA adapters using T4 RNA Ligase (NEB). Adapter primers were used for cDNA synthesis, followed by RNA degradation. Each cDNA library was ligated to a DNA adapter, used for barcoding and PCR enrichment of the library. Libraries were pooled and sequenced on the Illumina HiSeq 2000. ChIP-Seq: Cells were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads (Invitrogen) for 2-3 hour at 4°C, with constant agitation
Immunoprecipitated DNA was end repaired using the End-It DNA End-Repair Kit (Epicentre), extended using a Klenow fragment (3’-5’ exo) (NEB), and ligated to sequencing adapter oligos (Illumina). Each library was then PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300-600 was selected for sequencing on the Illumina HiSeq 2000.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

mES_Nanog_3p_2i.narrowPeak

Data processing

Base calling was performed with the standard Illumina pipeline 1.83
ChIP-Seq data was aligned to the mm9/NCBI37 reference genome using bwa version 0.5.7 (Li and Durbin, 2009) or with bowtie version 0.12.7 (Langmead et al., 2009) with default parameter settings. Subsequently, reads were filtered for duplicates and extended by 200bp. Visualization of read count data was performed by converting raw bam files to .tdf files using IGV tools (Thorvaldsdottir et al., 2012) and normalizing to 1 million reads. For the analysis of the ChIP-Seq datasets, we utilized the Irreproducible Discovery Rate (IDR) framework with a cutoff of 0.05 in combination with the MACS2 peak caller version 2.1 to identify peaks taking advantage of both replicates for each condition (except for OCT4). For MACS2 peak calling, we used an initial p-value cutoff of 0.01 and the mouse ESC whole cell extract (WCE) control library as background.
RNA-Seq raw sequence reads were aligned to mm9 using Tophat (Trapnell et al., 2009) and Bowtie version 0.12.7 (Langmead et al., 2009) with parameters set to default values except mate-inner-dist 300 mate-std-dev 500. Subsequently, duplicates were removed and cufflinks 2.2.1 (Grant et al., 2011) was used to conduct RNA expression quantification based on ensembl mm9 gene models. The –M option of cuffquant/cuffdiff was used to mask out reads mapping to rRNA genes, mitochondrial genes, and any transcripts less than 200 bp long. A pseudo-count of 1 was added to all FPKM values generated from cuffdiff before plotting. An FDR threshold of 0.1 was used for differential expression.
Genome_build: mm9
Supplementary_files_format_and_content: Processed data for ChIP-Seq is in ENCODE narrowPeak format. Processed gene expression data is in standard cuffdiff .tracking format.

Submission date

Aug 03, 2015

Last update date

May 15, 2019

Contact name

Michael Johannes Ziller

Organization name

Max Planck Institute of Psychiatry

Department

Translational Psychiatry

Lab

Ziller lab