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Sample GSM1400771 Query DataSets for GSM1400771
Status Public on Jul 08, 2014
Title MNase Sperm Unspun .5 Units
Sample type SRA
Source name Sperm, .5 units MNase, unspun
Organism Mus musculus
Characteristics strain/background: C57/B6J
age: 10 weeks
tissue: sperm
chromatin preparation: Micrococcal Nuclease
micrococcal nuclease units: 0.5
fraction: non-separated
antibody: none
Treatment protocol Sperm were isolated from the caudal epididymis and vas deferens of 10-week-old C57/6J mice by lacerating tissue in 1 mL of 37°C M2 medium (Sigma). Sperm were allowed to swim up for 1 hr, washed 1X in water, 1X PBS, crosslinked at 37°C with 1% formaldehyde for 15 min, quenched with 250mM glycine for 5 min and frozen in liquid nitrogen.
Growth protocol E14 ES cells were cultured on gelatin-coated dishes without feeder cells in standard media containing serum and LIF at 37°C with 5% CO2.
Extracted molecule genomic DNA
Extraction protocol Permeabilized ES and sperm nuclei were treated with 10 Units/10^6cells and 1 Unit/10^8cells of micrococcal nuclease (Worthington), respectively, for 5 min @ 37°C. Reaction was stopped with the addition of 10 mM EGTA on ice. For ES cells, nuclei were incubated for 4 hrs at 4°C with rotation, pelleted at 5000g for 5 min, and separated into supernatant and pellet (spun) or were treated the same without centrifugation and separation (unspun). After MNase digestion of sperm nuclei, digestion was centrifuged for 5000g for 5 min.
To characterize the entire range of MNase digestion products, we generated paired-end libraries of DNA fragments from both ES and sperm digestions and performed sequencing using Illumina HiSeq technology. Libraries were prepared as described in PMID: 22025700.
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina MiSeq
Description Non-Separated Fraction of DNA fragments resulting from Microccocal nuclease (.5 Units) digestion of sperm chromatin.
Data processing Removal of 3' adapter sequences using eautils 1.1.2.
Alignment of trimmed sequences to genome using Bowtie v2-2.2.2.
Creation of BigWig using standard scripts in Samtools v0.1.16 and bedGraphToBigWig (available at UCSC genome).
Tab-delimited txt files representing alignment of deepseq libraries to Transcriptional Start Site (TSS) in 20bp windows upstream and downstream of TSS 2000bp using the program SeqMiner v1.3.3e.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: BigWig, tab-delimited text files.
Submission date May 29, 2014
Last update date May 15, 2019
Contact name Benjamin R Carone
Organization name UMass Medical School
Department BMP
Street address 364 Plantation St
City Worcester
State/province Massachusetts
ZIP/Postal code 01605
Country USA
Platform ID GPL16417
Series (1)
GSE58101 High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm
BioSample SAMN02803889
SRA SRX555509

Supplementary file Size Download File type/resource
GSM1400771_MNase_Unspun_.5_135-165.txt.gz 611.5 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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