GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1182877

Query DataSets for GSM1182877

Status

Public on Nov 04, 2013

Title

dRF6: MEF input

Sample type

SRA

Source name

MEF input

Organism

Mus musculus

Characteristics

strain: F1: 129S1/CastEiJ x 129S1
cell type: immortalized MEFs
capture oligos: n/a
sequenced molecule: genomic DNA of input

Treatment protocol

d0 ES cells were cultured in the presence of LIF under adherent conditions. d7 ES cells were cultured without LIF under non-adherent conditions for 4 days and plated for an additional 3 days.

Growth protocol

Cells were cultured in regular fibroblast medium (1% Pen/Strep, 1% L-glutamine, high-glucose DMEM) with either 15% FBS (MEFs) or 30% FBS (ES cells)

Extracted molecule

genomic DNA

Extraction protocol

Nuclei, purified from cells crosslinked in 1% formaldehyde for 10 min, were further crosslinked with 3% formaldehyde for 30 min at room temperature. Sheared chromatin, with a median size around 3kb, was incubated overnight with a mixture of biotinylated capture oligonucleotides that were complementary to Xist RNA. The hybridized material was captured by streptavidin beads, and washed extensively before RNaseH elution. Enriched DNA was purified after formaldehyde crosslink reversal and subjected to library construction.
Genome DNA of inputs and Xist CHART enriched DNA samples were fragmented by sonication, and the sequencing libraries was constructed by first repairing DNA-ends, A-tailing, ligating to universal adapters, and amplifying for 12 cycles with indexed primer.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

Library strategy: CHART-Seq
Basecalls performed using Illumina software, version 1.8, except for dSP20b, which was performed using version 1.3
CHART-seq: Alignment was performed with Novoalign 3.00 (www.novocraft.com) using default parameters with modifications: -t 180 -h 180 180 -v 180. Uniquely aligned pairs were selected, discarding duplicate fragments. SPP was used to normalize each sample to its corresponding input accepting all tags and smoothing with 1000 kb windows sampled every 500bp. To normalize across data sets, each bedGraph was normalized to sum of all positive values on chr4.
RNA-seq: Adapter sequences were stripped from reads and identical reads discarded. Alignment was performed using tophat2 with the “b2-sensitive” preset and otherwise default parameters. After removal of PCR duplicates, all unique reads mapping to gene bodies were summed up for the cas, mus and comp tracks. Read numbers over genes in the allelic tracks were used to calculate skew (mus-cas/mus+cas) and genes skewed significantly (P < 0.01, cumulative binomial probability) in d7 ES cells and MEFs output to bedGraph.
Each dataset was aligned to variant CAST/EiJ and 129S1/SvJm genomes constructed using high quality polymorphisms to the C57/Bl6 reference genome (mm9 build). Pairs aligning to only one variant genome and pairs aligning better to one variant genome (in number of nucleotide edits to reference) than the other were retained (allele-specific), as were pairs aligning equally well (non-allele-specific).
Repetitive alignments, duplicate reads and low quality reads were removed. The resulting files were analyzed using SPP software. In this analysis all tags were included and the coverage generated with smoothing using 1kb bins every 500 bp to generate input-subtracted, normalized read densities. To account for different read depths across data sets, each coverage file was scaled using the total positive read density on an autosome (chr4) from the corresponding composite track (i.e., the mus, cas and comp tracks were all scaled using the same factor) and output to bedGraph.
CHIP-seq: Data from GSE36905 was aligned and processed as CHART-seq samples. Resulting coverage tracks (EZH2/K27me3) are linked directly to GSE48649 (bedGraphs).
Genome_build: mm9
Supplementary_files_format_and_content: All processed data files are in bedGraph format and represent either spp enrichment score for CHART-seq and CHIP-seq or skewed expression value ranging from -1 (all CAST/EiJ) to +1 (all 129S1) for RNA-seq

Submission date

Jul 09, 2013

Last update date

May 15, 2019

Contact name

Stefan F Pinter

E-mail(s)

spinter@uchc.edu

Organization name

University of Connecticut, UConn Health

Department

Genetics and Genome Sciences