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Sample GSM914028 Query DataSets for GSM914028
Status Public on Jan 28, 2013
Title Human Unmethylated Nucleosome Sample R2
Sample type SRA
 
Source name Human Unmethylated Nucleosome
Organism Homo sapiens
Characteristics methylation status: Unmethylated
bac clone source: RP11-89D11, RP11-440N18, RP11-980A6
Treatment protocol The CpGs in equal molar mixtures of BAC DNAs were methylated by treatment with CpG Methyltransferase (M.SssI) from New England Biolabs (2 Units of Methyltransferase / µg DNA overnight at 30°C), which is claimed to methylate all cytosine residues in CpG dinucleotides. Completion of CpG methylation was verified by resistance to digestion with methylation-specific endonucleases (BstUI & HhaI) with subsequent analysis on an agarose gel.
Growth protocol BAC clones on LB agar stabs were obtained from the BACPAC Resources Center and were grown according to instructions provided by the supplier. BAC DNAs were isolated by standard protocols and sheared 10 times through a 26 gauge needle. Equal molar mixtures of the BAC DNAs were then prepared.
Extracted molecule genomic DNA
Extraction protocol Equal molar mixtures of the BAC DNAs were reconstituted into nucleosomes under selective pressure for nucleosome-favoring sequences. The unmethylated or methylated DNA was mixed with core histones (isolated from H1/H5-stripped chicken erythrocyte nucleosomes) in 2.0 M NaCl at a DNA-histone mass ratio of 5:1 with 60 µg of DNA in a volume of 150 µl. Nucleosome reconstitution was carried out by gradient dialysis (2.0 M NaCl to 0.01 M NaCl) over a period of 16 hours at 4°C. Samples (30 µg DNA in 2 parallel reactions) were digested with micrococcal nuclease (MNase) using 1 Worthington unit at 37°C for 5 minutes with 1 mM CaCl2 in a volume of 200 µl. The samples were then deproteinized, protected fragments were extracted, and mononucleosome-length DNA was isolated by gel electrophoresis on a 5% polyacrylamide gel. For controls, naked DNA was fragmented to a length of ~150 bp with a Covaris sonicator, and naked DNA (30 µg) was digested separately with MNase using 0.3 Worthington units at 37°C for 5 minutes with 1 mM CaCl2 in a volume of 120 µl. These controls were then size-selected on a 5% polyacrylamide gel (between 140 and 160 bp). It is important to note that all nucleosome samples were biological, not technical, replicates and were produced on different days. The unmethylated/methylated nucleosome and control samples for both human and mouse were submitted to the Purdue Genomics Core Facility where these 14 samples were end-repaired, barcoded, nick translated, amplified by 7 cycles of PCR, and simultaneously sequenced with the SOLiD paired-end sequencing technology in a single run on a quarter slide.
 
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model AB SOLiD 4 System
 
Description Sample 2
Data processing Reads were mapped to the reference sequence with the Bioscope (bioscope-v1.3-rBS130_50843_20101020105719) software. The human reference sequence consisted of 3 BACs: RP11-89D11 (hp53), RP11-440N18 (hmyc), and RP11-980A6 (halb). The mouse reference sequence consisted of 3 BACs: RP23-51O13 (mp53), RP23-302D24 (mmyc), and RP23-67F18 (malb). Information in the output SAM files was used to identify and localize the midpoints of properly-paired reads with desired insert lengths (141-155 bp). Only properly-paired reads with the following SAM flags were utilized: 83, 99, 147, 163, 1107, 1123, 1171, and 1187. We defined the midpoint of a paired-end read by adding int((insert length - 1) / 2) to the left-most coordinate of the paired-end read (int = integer truncation). Therefore, if a read insert was 147 bp in length, the midpoint would be located 73 bp downstream from the left-most coordinate. The number of properly-paired reads with desired insert lengths at midpoints was then determined at each position for each individual library, and these processed data are provided in text files for the 7 human and 7 mouse samples.

The properly-paired read counts in the 3 unmethylated and 2 methylated human nucleosome libraries at each position were normalized by the average number of read midpoints per base pair, which is equal to the total number of reads in a library divided by the combined lengths of the BACs. To account for midpoint discrepancies, the counts were converted to 3 bp sliding window sums. After normalization and conversion to 3 bp sums, combined unmethylated and methylated libraries were produced by adding the counts at each position together and then dividing by three and two, which are the number of unmethylated and methylated libraries, respectively. The same procedure was done for the mouse data. These combined normalized unmethylated and methylated libraries in 3 bp sums are available in text files for both human and mouse [on Series records].

11,448 human nucleosome positions were then generated by non-specific filtering and a peak finding algorithm, using the human combined unmethylated and methylated nucleosome libraries (See Methods in Paper). 13,741 mouse nucleosome positions were also generated in the same manner, using the mouse combined unmethylated and methylated nucleosome libraries. Additionally, for the mouse unmethylated and methylated MNase controls, 12,884 positions were derived. For these 3 datasets, the BAC, the BAC coordinate, the numbers of unmethylated and methylated reads, the fractional difference, and the numbers of CGs in the insert (made to be 147 bp surrounding the midpoint) are given for each position in txt files. For each of these positions, the fractional difference between the unmethylated and methylated libraries was computed by dividing the absolute difference between the unmethylated and methylated peaks by the larger peak of the two and multiplying by -1 if the larger peak was from the unmethylated library. Additionally, for the human and mouse nucleosome libraries, the adjusted p-values that were assigned by DESeq are given, and for just the human nucleosome libraries, the base mean of the five libraries as well as translational scores for each position are also provided.

For differential nucleosome positioning analysis, the statistical software package DESeq was utilized, which is implemented in R and distributed by the Bioconductor project. DESeq fits count data to a negative binomial model and is typically used to test for differential gene expression in RNA-Seq datasets with small sample sizes. For the human data, the number of normalized reads in 3 bp sums from the three individual unmethylated and the two individual methylated replicates at the 11,448 nucleosome midpoint positions were used as the human count dataset input into DESeq. For the mouse data, the number of normalized reads in 3 bp sums from the two individual unmethylated and the two individual methylated replicates at the 13,741 nucleosome midpoint positions were used as the mouse count dataset input into DESeq. These two count datasets are presented in text files. Note that these numbers are required to be rounded to the nearest integer for analysis by DESeq and that the size factors should be set to 1 since the data are already normalized.

Genome_build: hg19, mm9

Supplementary_files_format_and_content: There are 23 text files, and their content is described in the data processing steps.
 
Submission date Apr 12, 2012
Last update date May 15, 2019
Contact name Clayton Kenneth Collings
E-mail(s) ccolling@purdue.edu
Phone 812-593-2314
Organization name Purdue University
Department Biological Sciences
Lab John N. Anderson
Street address 915 W. State Street
City West Lafayette
State/province Indiana
ZIP/Postal code 47907
Country USA
 
Platform ID GPL13393
Series (1)
GSE37224 Effects of DNA Methylation on Nucleosome Stability
Relations
SRA SRX141235
BioSample SAMN00854935

Supplementary file Size Download File type/resource
GSM914028_human_unmethylated_nucleosome_R2.txt.gz 1.5 Mb (ftp)(http) TXT
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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