GSM1535345: whole_run2; Mus musculus; RNA-Seq
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.
GEO Accession
GSM1535345