Antigen

Antigen Class

ATAC-Seq

Antigen

ATAC-Seq

Cell type

Cell type Class

Gonad

Cell type

Spermatids

MeSH Description

Male germ cells derived from the haploid secondary SPERMATOCYTES. Without further division, spermatids undergo structural changes and give rise to SPERMATOZOA.

Sample

source_name

testes

strain

C57BL6 GCN5+/+

tissue

testes

Stage

Meiotic Spermatid

replicate

1

Sequenced DNA Library

library_strategy

ATAC-seq

library_source

GENOMIC

library_selection

other

library_construction_protocol

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. ATAC-seq was performed as previously described(Buenrostro et al., 2013), with slight modifications(Sammons et al., 2015). Cells were washed with cold PBS, aliquoted (M and Sp: 100k cells/rxn, RS and ES: 200k cells), and centrifuged at 500xg for 10min at 4C in swinging bucket centrifuge. Following lysis in standard ATAC lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, and 0.1% IGEPAL CA-630), recovered nuclei were tagmented with 2.5U and 5.0U of Tn5 in parallel reactions. Libraries were amplified for 12 PCR cycles and quantified by KAPA. Libraries were prepared from two independent STAPUT collections for SV129E cells and 1 STAPUT for Gcn5Ctrl and Gcn5cKO. Sperm libraries were prepared from individual animals. All libraries were sequenced on the Illumina NextSeq with paired-end sequencing for 150 cycles.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

62202429

Reads aligned (%)

53.2

Duplicates removed (%)

23.4

Number of peaks

22014 (qval < 1E-05)

mm9

Number of total reads

62202429

Reads aligned (%)

53.1

Duplicates removed (%)

23.4

Number of peaks

21910 (qval < 1E-05)