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Sample GSM883648

Query DataSets for GSM883648

Status

Public on May 24, 2013

Title

NPC_input DNA

Sample type

SRA

Source name

Neural progenitor cells

Organism

Mus musculus

Characteristics

cell type: ES-derived neural progenitor cells
strain: 129SvJae x C57BL/6
gender: male
chip antibody: none

Treatment protocol

V6.5 ES cells were differentiated into neural progenitor cells (NPCs) using established techniques (Meissner et al., 2008; Mikkelsen et al., 2007; Okabe et al., 1996). Briefly, after expansion, ES cells were trypsinized and cultured in bacterial dishes for 4 days in the absence of LIF to promote embryoid body formation. Embryoid bodies were then plated on tissue-culture plastic dishes and cultured in serum-free, defined media (ITSFn). After 5-7 days selection in ITSFn, adherent cells were trypsinized, triturated to a single cell suspension, and re-plated on tissue culture dishes coated with 15 ug/ml poly-L-ornithine (Sigma) and 1 ug/ml human plasma fibronectin (Gibco). NPCs were further propagated for 2-4 days in DMEM/F12 supplemented with 25 ug/ml insulin, 50 ug/ml human APO transferrin, 30 nM sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine (Sigma), 1 ug/ml laminin (Sigma), 10 ng/ml Fgf2 (R&D Systems), and 1x penicillin/streptomycin. NPCs were ~60-75% confluent at the time of fixation with 1% formaldehyde before downstream assay.

Growth protocol

Murine V6.5 ES cells were expanded on Mitomycin-C-inactivated MEF feeder layers in pluripotent media supplemented with LIF. After initial expansion, ES cells were passaged 2-3 times on 0.1% gelatin to remove contaminating feeder cells.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin immunoprecipitation (ChIP) was performed as previously described with minor modifications (Kagey et al., 2010). Briefly, 50x106 NPCs were lysed and chromatin was sonicated with a Branson Sonifier 250 for 30 x 12 sec pulses. Chromatin was pre-cleared with Protein A Sepharose beads and then immunoprecipitated with approximately 10 μg of the appropriate antibody coupled to Protein A sepharose beads. For Smc1a ChIP using Bethyl Laboratories (A300-055A) affinity purified rabbit polyclonal antibody, beads were washed 1X with LB3, 1X with 20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100, 1X with 10mM Tris-HCl pH8, 250nM LiCl, 2mM EDTA, 1% NP40 and 1X with TE containing 50 mM NaCl. For CTCF ChIP using an Upstate 07-729 rabbit polyclonal antibody, beads were washed 7 times with RIPA buffer and 1X with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C for 15 min with occasional vortexing. Cross-links were reversed by overnight incubation at 65°C. Whole cell extract DNA reserved from the sonication step was also treated for crosslink reversal. ChIP-seq libraries were prepared for sequencing as recommended by Illumina/Solexa.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

ChIP-seq

Data processing

Sequences were aligned to NCBI Build 37 (UCSC mm9) using default parameters (-v1 -m1) in Bowtie. Only sequences that mapped uniquely to the genome were used for further analysis. Model-based Analysis for ChIP-Sequencing (MACS) was used for peak calling (http://liulab.dfci.harvard.edu/MACS/00README.html) (Zhang et al., 2008). Default parameters were used with a p-value cutoff of P<1E-8. A background whole cell extract input control was used to assess significance.

Submission date

Mar 01, 2012

Last update date

May 15, 2019

Contact name

Jennifer Elizabeth Phillips-Cremins

E-mail(s)

Jennifer.Phillips-Cremins@umassmed.edu

Organization name

University of Massachusetts Medical School and Emory University

Department

Program in Systems Biology and Department of Biology