GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1535344

Query DataSets for GSM1535344

Status

Public on Jul 08, 2015

Title

nuc2_run2

Sample type

SRA

Source name

sucrose fractionated mouse sperm nuclei RNA

Organism

Mus musculus

Characteristics

strain: C57BL/6 transgenic line HP3.1
cell type: sperm
cell fraction: sucrose-fractionated sperm nuclei
developmental stage: adult

Extracted molecule

total RNA

Extraction protocol

Mature spermatozoa from transgenic line HP3.1 were isolated from cauda epididymides and vas deferens harvested from individual four month old transgenic mice on ice into 50 mM Tris-HCl, pH 7.4, buffer. The cells were washed twice following filtration through an 80 micron mesh, resuspended in 0.5 mL 50 mM Tris-HCl, 7.4, buffer and subjected to sonication with a TekMar TM-50 sonic disruptor (TekMar, Cincinnati, OH) at 70% maximum output for two minutes on ice to separate heads from tails. Separation was confirmed by light microscopy. The sperm solution containing 1 – 5 x 107 cells was diluted to a volume of 7.5 mL in 1 M sucrose buffered with 50 mM Tris-HCl, pH 7.4, buffer containing 5 mM MgCl2. A triple-step gradient was prepared by overlaying diluted samples onto cushions consisting of 2 M sucrose buffered with 50 mM Tris-HCl pH 7.4 containing 5 mM MgCl2 and 0.45 g/mL CsCl buffered with 25 mM Tris-HCl, pH 7.4, buffer containing 0.5% Triton X-100. Nuclei were recovered by ultracentrifugation at 75,600 x g for 45 minutes at 4° C and subsequently washed twice with 25 mM Tris-HCl, pH 7.4, buffer containing 0.5% Triton X-100. Following resuspension in 0.5 mL RLT buffer (Qiagen, Valencia, CA) supplemented with 1.5% β-mercaptoethanol (Amresco, Rockford, IL), 0.2 mm stainless steel beads were added to the samples and nuclei were lyzed with the Disruptor Genie (Scientific Industries, Inc., Bohemia, NY). After the addition of an equal volume of Qiazol (Qiagen) nucleic acids were recovered using the RNeasy system (Qiagen). Whole cell sperm RNAs were equivalently extracted following visual confirmation of the absence of contaminating cell-types omitting sonication and ultracentrifugation. Extracted total RNAs were DNased (Turbo Dnase, Ambion, Grand Island, NY) and subsequently subjected to RT-PCR with intron-spanning primers to Prm2 (Foward 5'-GCACCATGGTTCGCTACCG-3', Reverse 5'- GTGGCCTCACATGATGTTGCT-3'; 31).
Stock synthetic spike-in ERCC RNAs (Invitrogen, Carlsbad, CA) were diluted 1:10,000 and pooled with total sperm RNAs prior to reverse transcription and amplification using the Seq-plex system (Sigma, St. Louis, MO). Following universal priming site removal pre-amplified cDNA libraries were subjected to sequencing library construction (DNA Ultra-Low, NEB, Ipswich, MA) followed by 50 cycles of paired-end sequencing on the Illumina Hi-Seq 2500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

sperm nuclei 2
processed data file: nuc2_subcount.txt
processed data file: unadjusted RPKMs.txt

Data processing

Illumina Casava v 1.8.2 software used for basecalling and demultiplexing
Sequencing reads were aligned to the mouse genome assembly Mm10 with Tophat2 (version 2.0.12) using the following parameters: tophat2 -r 30 --mate-std-dev 50 --no-coverage-search -G genes.gtf.
Novel transcript structures were assembled using Cufflinks (version 2.1.1). Assembled transcripts exceeding twice the average fragment length (135 bp) were combined with UCSC gene annotations (Mm10). Transcript abundance was calculated using uniquely aligned sequencing reads and the Samtools (version 0.1.19; 34) and Bedtools (version v2.19.1-2) suites.
Coverage of the most abundant isoform of each transcript was calculated in reads aligned per kilobases mapped (RPKM) using Bedtools.
Differential enrichment and subtractive differential enrichment analyzes were performed with the HT-Seq and DEseq2 software packages. The latter required randomly subsampling uniquely aligned reads to equivalent levels using Samtools prior to alignment with TopHat2. Subsequently nuclei naïve count values from HT-Seq were averaged, rounded to whole integers and subtracted from corresponding whole cell naïve values. Negative adjusted whole cell counts were assumed to be zero. Differential enrichment analysis was carried out using DESeq2 as before.
Genome_build: Mm10
Supplementary_files_format_and_content: unadjusted RPKM values, tab-delimited text files (Ht-seq counts)

Submission date

Oct 30, 2014

Last update date

May 15, 2019

Contact name

Graham Johnson

Organization name

WSUSOM

Department

CMMG

Lab

Krawetz