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Sample GSM883651

Query DataSets for GSM883651

Status

Public on May 24, 2013

Title

NPC_Rep1_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 (http://bowtie-bio.sourceforge.net/index.shtml) (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

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