GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1223553

Query DataSets for GSM1223553

Status

Public on May 29, 2014

Title

mouse_tgc_h3k4me3 (paired-end)

Sample type

SRA

Source name

Trophoblast giant cells - cultured

Organism

Mus musculus

Characteristics

strain: 129/Sv
chip antibody: H3K4me3 (ActiveMotif, 39159, lot 1609004)
cell type: cultured trophoblast giant cells

Treatment protocol

TSCs were differentiated into trophoblast giant cells (TGCs) by using differentiation media, which consisted of TS media without FGF4, Activin, and Heparin, with the addition of retinoic acid dissolved in ethanol to the media to a final concentration of 5 μM. TSCs were differentiated for five days, replacing media every other day, before harvesting.

Growth protocol

Mouse trophoblast stem cells (TSCs) were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004).

Extracted molecule

genomic DNA

Extraction protocol

Cells were trypsinized for 4 minutes and prepared using the ChIP kit (Millipore). Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Catalog # FC-102-1001). 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 (3' to 5' 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 200~400 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 Illumina HiSeq following the manufacturer's protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

ChIP-Seq

Data processing

Basecalls performed using CASAVA version 1.5
ChIP-seq reads were aligned to mm9 using BWA 0.5.9 (-q 10 -I), and filtered using samtools view (-q 10) to remove multiply mapping reads.
Individual alignments were merged using samtools merge, then peaks were called using MACS v2.0.10 with default settings
genome build: = mm9
supplementary files format and content: ChIP-Seq bed files contain peaks called by MACS v2.0.10

Submission date

Sep 03, 2013

Last update date

May 15, 2019

Contact name

Roberta L Hannibal

E-mail(s)

robertah@stanford.edu

Organization name

Stanford University

Department

Genetics