GSM3955839
GSM3955839: Meiotic Spermatid 1U Mnase Replicate 1; Mus musculus; MNase-Seq
Sexually mature, adult male SV129 mice (8-12wks old, Charles River Laboratories) were humanely euthanized by CO2 asphyxiation according to University of Pennsylvania Institutional Animal Care and Use Committee guidelines.  Immature male germ cells were collected from testes by STAPUT velocity sedimentation as previously optimized, described, and validated by our laboratory (Bryant et al., 2013).  Briefly, for each STAPUT collection testes from 11 adult SV129 mice were dissociated to a single cell suspension with sequential incubation of collagenase (0.9 mg/ml) and trypsin (0.6 mg/ml) with DNase (0.1 µg/ml) in KREBs buffer.  Testes cells were subsequently resuspended in 0.5% BSA and slowly loaded onto a 2-4% BSA gradient.  Cells were allowed to sediment for 105 min prior to fraction collection.  Cell aliquots were stained with DAPI and assessed by fluorescent microscopy for meiotic (M), round spermatid (RS), and elongating/condensing spermatid (ES) fractions.  Cell fractions with at least 85% purity for each stage were pooled, snap frozen, and stored at -80C for further processing.  Mature sperm were collected from the cauda epididymis and incubated in somatic cell lysis buffer (0.1% SDS, 0.5% Triton-X-100) for 15min to remove any non-sperm contaminants.  For each collection the sperm of 11 animals were pooled, counted, and subsequently snap frozen and stored at -80C until further processing. SV129 M (105) and RS (2x105) cells were digested with MNase as previously described {Amour:1kr} with slight modifications.  Digestions were carried out with 0U, 0.5U, 1U, or 2U of MNase (Roche) for 7.5min at 37C.  The resulting DNA was phenol:chloroform purified, quantified (Qubit), and tested for quality and correct nucleosomal size distribution with Bioanalzyer DNA 1000 analysis (Agilent). Sequencing libraries were generated with the NEB Next Ultra II DNA Library Prep Kit for Illumina as described by manufacture protocol.  Libraries were sequenced on the Illumina NextSeq with paired-end sequencing for 75 cycles.
GEO
GSM3955839