Antigen

Antigen Class

ATAC-Seq

Antigen

ATAC-Seq

Cell type

Cell type Class

Gonad

Cell type

Spermatogonia

MeSH Description

Euploid male germ cells of an early stage of SPERMATOGENESIS, derived from prespermatogonia. With the onset of puberty, spermatogonia at the basement membrane of the seminiferous tubule proliferate by mitotic then meiotic divisions and give rise to the haploid SPERMATOCYTES.

Sample

source_name

Thy1+ spermatogonia

strain

C57BL/6

tissue

testis

age

P7

genotype

Scml2-KO

biowardrobe ides

3842

Sequenced DNA Library

library_strategy

ATAC-seq

library_source

GENOMIC

library_selection

other

library_construction_protocol

The ATAC-seq libraries were prepared as previously described (Buenrostro et al. 2013). Around 500,000 of Thy1+ or c-Kit+ spermatogonia, 250,000 of pachytene spermatocytes, and 1,000,000 of round spermatids were used. Briefly, samples were lysed in 50 μl of lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40). Immediately after lysis, nuclei were spun at 500g for 5 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposes and tagmentation buffer at 37 °C for 30 min (Illumina). After the tagmentation, the transposed DNA was purified by MinElute kit (Qiagen). PCR was performed to amplify the library using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min; following by 72 °C 5 min. qPCR was used to estimate of the number of additional cycles needed to generate products at 25% saturation. Typically, two to five additional PCR cycles were added to the initial set of five cycles. The library was purified by AMPure XP beads (Beckman). Size selection of library pools were achieved by running on agrose gel electrophoresis and excising gel slices in the 250- to 500-bp size range. The pool purified from the gel slices was analyzed on an Agilent Bioanalyzer, and 75-bp single-read sequencing was performed using an Illumina HiSeq 2500 platform according to standard oper¬ating procedures.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

64355369

Reads aligned (%)

75.5

Duplicates removed (%)

62.9

Number of peaks

34139 (qval < 1E-05)

mm9

Number of total reads

64355369

Reads aligned (%)

75.5

Duplicates removed (%)

62.9

Number of peaks

34150 (qval < 1E-05)