Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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

P6 ID4-EGFP+ spermatogonia from mouse testes

strain

LT-11B6 (C57BL/6)

cell population

TSPAN8-low subpopulation of P6 ID4-EGFP+

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Sorted P6 ID4-EGFP+ spermatogonia and subpopulations defined by antibody labeling for TSPAN8 were used for Reduced-representation bisulfite sequencing (RRBS) analysis of methylome or ChIP-seq analysis of post-translational modifications of histones (H3K4me3, H3K27me3 and H3K27Ac). For RRBS, snap-frozen cell pellets from five independent sorts of TSPAN8-High and TSPAN8-Low ID4-EGFP+ spermatogonia were used for genomic DNA isolation and RRBS was performed using the Methyl-MidiSeq service by Zymo Research (Irvine, CA). For ChIP-seq, snap-frozen cell pellets from eight independent sorts of the TSPAN8-High and TSPAN8-Low subpopulations were subjected to low-input ChIP-seq service by Active Motif (Carlsbad, CA) using proprietary approaches. Briefly, native (unfixed) chromatin was isolated, quantified, and 9000 (H3K4me3) to 34000 (H3K27me3 and H3K27Ac) cell equivalents of chromatin (6.6pg/cell) were used for each ChIP reaction using antibodies against H3K4me3 (Active Motif 39159), H3K27Ac (Active Motif 39133) or H3K27me3 (Millipore, 07-449). ChIP'd genomic DNA or genomic DNA from residual chromatin were subsequently used for sequencing. For RRBS, libraries were prepared from 300 ng of genomic DNA digested with the BfaI, MseI, and MspI restriction enzymes and the fragments produced were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine. Adapter-ligated fragments were filled in and 3’-terminal-A extended, then purified using the Zymo Research (ZR) DNA Clean & Concentrator – 5 kit (Cat#: D4003). Bisulfite treatment of the fragments was done using the EZ DNA Methylation – Lightning kit (ZR, Cat#: D5030). PCR was performed and the size and concentration of the fragments were confirmed on the Agilent 2200 TapeStation, then sequenced on the Illumina Hiseq 2500 with PE50 parameters. For ChIP-seq, ChIP and input samples were prepared for amplification by converting overhangs into phosphorylated blunt ends, 3’ adenylation, ligation of Illumina adaptors and library size selection (175-225 bp) on an agarose gel. Adaptor-ligated libraries were then amplified for 18 cycles and resulting DNAs were purified, quantified, and tested by qPCR to assess quality of amplification reactions. Amplified DNA libraries subjected to Illumina sequencing with HiSeq2000 instrument (SE50).

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

46227896

Reads aligned (%)

33.4

Duplicates removed (%)

54.7

Number of peaks

983 (qval < 1E-05)

mm9

Number of total reads

46227896

Reads aligned (%)

33.3

Duplicates removed (%)

54.9

Number of peaks

965 (qval < 1E-05)