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Sample GSM883652 Query DataSets for GSM883652
Status Public on May 24, 2013
Title NPC_Rep2_5C_library
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 3C templates were generated using HindIII as previously described (Dekker et al., 2002; van Berkum and Dekker, 2009). 5C libraries were generated from 3C templates using an alternating primer design across 1-2 Mb regions around Oct4, Nanog, Sox2, Nestin, Olig1-Olig2, Klf4, and a gene desert negative control (described previously Dostie and Dekker, 2007; Dostie et al., 2006; van Berkum and Dekker, 2009).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
Description 3C and 5C
Data processing Reads were aligned to a pseudo-genome consisting of all 5C primers using Bowtie ( (Langmead, 2010). To account for poor quality reads, sequences were required to have only one unique alignment and 5 and 3 bases were trimmed from the 5’ and 3’ ends of the read, respectively. Interactions were counted when both paired end reads could be uniquely mapped to the 5C primer pseudo-genome. Only interactions between forward-reverse primer pairs were tallied as a true count, as forward-forward or reverse-reverse primer pairs represent an artifact in the 5C procedure.
To account for bias intrinsic to all 3C-based methods, as well as to 5C specifically, we developed a probabilistic model that simultaneously captures the non-biological contribution of specific primers and the distance-dependent background level of non-specific chromatin interactions (Imakaev et al., 2012; Yaffe and Tanay, 2011). For each region, model parameters describing the expected value and signal-dependent variance of each interaction under a normal-lognormal distribution were learned using stochastic gradient descent. Using the learned parameters, empirical p-values were computed under this distribution via Monte Carlo simulation for each observed interaction count. Interaction scores were derived using the inverse cumulative density function for a standard normal distribution. The resulting p-values and derived interactions scores are directly comparable within and between experiments and allow for robust detection of fragment-to-fragment looping interactions that are significant above the expected background signal.
The processed data file contains the following information for each forward-reverse primer combination possible in cis: “Region” (5C Region), “Fprimer” (Forward primer ID), “Rprimer” (Reverse primer ID), “Distance” (mid-to-mid distance between fragments represented by forward and reverse primers), “Raw” (raw number of reads for a particular forward-reverse primer combination), “_.Predicted” (expected number of reads modeled from learned parameters for a particular forward-reverse primer combination), “_.PE” (primer-corrected counts for a particular forward-reverse primer combination), “_.DD” (primer-corrected and distance-corrected counts for a particular forward-reverse primer combination), “_.Pvalue” (pvalue assigning significance to the counts for a particular forward-reverse primer combination after comparison to the expected distribution of counts at that distance scale), and “_.InteractionScore” (for each forward-reverse primer combination, interaction scores were derived from pvalues using the inverse cumulative density function for a standard normal distribution). Processed data file includes information for 2 replicates of ES cells and 2 replicates of ES-derived NPCs (ES_Rep1, ES_Rep2, NPC_Rep1, and NPC_Rep2).
Submission date Mar 01, 2012
Last update date May 15, 2019
Contact name Jennifer Elizabeth Phillips-Cremins
Organization name University of Massachusetts Medical School and Emory University
Department Program in Systems Biology and Department of Biology
Lab Job Dekker Laboratory and Victor Corces Laboratory
Street address 364 Plantation Street
City Worcester
State/province MA
ZIP/Postal code 01605
Country USA
Platform ID GPL13112
Series (1)
GSE36203 Architectural protein subclasses shape 3-D organization of genomes during lineage commitment
SRA SRX125279
BioSample SAMN00794618

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

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