|
Status |
Public on Sep 28, 2010 |
Title |
MaleGH_DNase_rep2 |
Sample type |
SRA |
|
|
Source name |
Liver tissue
|
Organism |
Mus musculus |
Characteristics |
strain: ICR sex: Male tissue: liver animal treatment: continuous growth hormone treatment sample treatment: DNase digestion average size (bp): 269 size distribution in cv (%): 40.7 assayed molecule: chromatin
|
Treatment protocol |
Continuous GH treatment of 7-week male mice was achieved using Alzet model 1007D micro-osmotic pumps (Durect Corporation, Cupertino, CA) implanted s.c. under ketamine and xylazine anesthesia delivering recombinant rat GH (obtained from Dr. Arieh Gertler, Protein Laboratories Rehovot, Ltd, Rehovot, Israel) at 20 ng rat GH/hr/gram body weight for 7 days (Holloway et al. 2006). RNA was then extracted from individual livers using Trizol reagent (Invitrogen) followed by reverse transcription using 1 ug total RNA and a High Capacity Reverse Transcription Kit (Applied Biosystems).
|
Growth protocol |
Adult male and female ICR mice were purchased from Taconic, Inc. or Charles River Laboratories, and were housed in the Boston University Laboratory of Animal Care Facility in accordance with approved protocols. Livers were collected from 8-wk old mice euthanized by CO2 asphyxiation followed by cervical dislocation.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Sucrose step gradient ultracentrifugation was performed to isolate small DNA fragments (< 1.5 kb) from both digested nuclei and control samples. Fractions that primarily contain fragments <1.5 kb (typically fraction 7) were purified on Qiagen columns (Cat. No. 28704) and assayed by qPCR using primers designed to amplify known hypersensitive regions (non-sex specific or sex-specific). Samples that showed strong enrichment of DNase I hypersensitive fragments and that showed strong sex differences were used for Illumina sequencing. For each biological replicate of pooled nuclei, sequencing was carried out using a total of ~ 30 ng DNA, pooled from at least 3 independent batches of DNase digested nuclei, to minimize the impact of inter-sample variability in DNase digestion. Sequences of DNase I-released DNA fragments were generated by an Illumina Genome Analyzer platform. Briefly, about 20 ng of DNA was subjected to end repair, adaptor ligation, and PCR enrichment using an Illumina sample preparation kit following manufacturer’s recommendation. A DNA library in the range of 200 to 300 bp was size-purified by gel electrophoresis. The concentration of properly ligated samples was estimated by qPCR, followed by cluster generation and sequencing. The sequencing reads were aligned to mouse genome mm9 by Eland extended with a maximum of 2 mismatch allowed in the first 25 bp.
|
|
|
Library strategy |
DNase-Hypersensitivity |
Library source |
genomic |
Library selection |
DNAse |
Instrument model |
Illumina Genome Analyzer II |
|
|
Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse (July 2007) genome using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained. Peaks: Peak detection was performed with the PeakSeq algorithm (Rozowsky et al, Nature Biotechnol 2009)
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|
|
Submission date |
May 11, 2010 |
Last update date |
May 15, 2019 |
Contact name |
David J. Waxman |
E-mail(s) |
djw@bu.edu
|
Organization name |
Boston University
|
Department |
Department of Biology and Bioinformatics Program
|
Street address |
5 Cummington Mall
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02215 |
Country |
USA |
|
|
Platform ID |
GPL9250 |
Series (1) |
GSE21777 |
Unbiased, Genome-wide in vivo Mapping of Transcriptional Regulatory Elements Reveals Sex Differences in Chromatin Structure Associated with Sex-specific Liver Gene Expression |
|
Relations |
SRA |
SRX020264 |
BioSample |
SAMN00013384 |