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Sample GSM352184 Query DataSets for GSM352184
Status Public on Dec 18, 2008
Title MNase-digested DNA from HeLa S3 cells
Sample type SRA
 
Source name MNase-digested DNA
Organism Homo sapiens
Characteristics HeLa S3 cells (ATCC/Snyder Lab)
Growth protocol HeLaS3 cells were grown in Minimum Essential Medium modified for Suspension (SMEM, Invitrogen), supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 5 x10^5 cells/ml.
Extracted molecule genomic DNA
Extraction protocol HeLa S3 cells were resuspended in MNase buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40, and 1 mM DTT) and treated with 50 units of micrococcal nuclease (USB) at 37°C for 1 h. The samples were treated with proteinase K for 2 h at 37°C, extracted twice with phenol-chloroform, and subjected to ethanol precipitation. After centrifugation, the samples were treated with RNase A (Qiagen) and centrifuged through G50 sephadex spin columns. Each sample was treated with 30 units of calf intestinal alkaline phosphatase (NEB) for 2 h at 37°C to remove 3' phosphate groups left by the MNase. After a second ethanol precipitation, the samples were treated with 30 units T4 polynucleotide kinase (NEB). DNA samples were run though Qiagen MinElute PCR columns, eluted with 15 μl of Qiagen buffer EB and size-selected (100-200 bp) on 2% agarose E-gels (Invitrogen). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 ~250 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 Genome Analyzer following the manufacturer's protocols.
 
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina Genome Analyzer II
 
Description size selected, MNase-digested genomic DNA from HeLa S3 cells
size selected for 100-200 bp
Data processing Raw data from the Illumina Genome Analyzer and Illumina Genome Analyzer II were analyzed with Illumina’s Firecrest, Bustard and GERALD modules for image analysis, basecalling and run metrics respectively. Replicates were combined for each sample. All reads were aligned against human genome build hg18. The PolII, Sono-Seq (large fragment, small fragment, and interferon-gamma treated), and normal IgG were scored relative to naked DNA using PeakSeq (see "Peak-Seq - Systematic Scoring of ChIP-Seq Experiments Relative to Controls by Rozowsky et al. Nature Biotechnology. In press). Signal files were created using a 200 bp sliding window for all samples other than Sono-Seq/Input DNA-Large Fragment (575 bp). Two peak lists were created for each sample: an unfiltered list of all hits with p-value < 0.05 and a subset of this list that was filtered based upon sample tag count and enrichment versus naked DNA. Cutoff values were scaled for each sample relative to the number of reads. For PolII, normal mouse IgG, and Sono-Seq/Input DNA (small fragments), we required all peaks to have at least 20 sample tags and an enrichment of at least 5.0 versus the naked DNA reference. For Sono-Seq (interferon-gamma stimulated), cutoffs of 16 sample tags and 4.0x enrichment were used. For Sono-Seq (large fragments), cutoffs of 13 sample tags and 3.0x enrichment were used. MNase-digested DNA was not scored.
 
Submission date Dec 17, 2008
Last update date May 15, 2019
Contact name Raymond K Auerbach
Organization name Yale University
Street address 266 Whitney Ave
City New Haven
State/province CT
ZIP/Postal code 06511
Country USA
 
Platform ID GPL9115
Series (1)
GSE14022 Mapping Novel Chromatin Regions Using Sono-Seq
Relations
SRA SRX012411
BioSample SAMN00004743

Supplementary file Size Download File type/resource
GSM352184_MNase_from_HeLa_eland_results_rep1_laneA_FC305KG_20080624_s_1.txt.gz 174.2 Mb (ftp)(http) TXT
GSM352184_MNase_from_HeLa_eland_results_rep1_laneB_FC305NC_20080703_s_1.txt.gz 240.5 Mb (ftp)(http) TXT
GSM352184_MNase_from_HeLa_eland_results_rep2_laneA_FC305NC_20080703_s_8.txt.gz 264.7 Mb (ftp)(http) TXT
GSM352184_MNase_from_HeLa_eland_results_rep2_laneB_FC3082F_20080709_s_8.txt.gz 160.7 Mb (ftp)(http) TXT
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