ZBTB43 eliminates Z-DNA in mouse prospermatogonia
ZBTB43 ChIP-seq Purification of fetal male germ cells and somatic cells of the testis: To obtain fetuses for germ cell purification, C57Bl/6J females were crossed with OG2 (Szabo et al., 2002) males (Oct4-EGFPTg/Tg; Zbtb43+/+). Testicles were dissected from the resulting male fetuses at 15.5 days post coitum (dpc), and were dissociated to single cell suspension using trypsin digestion and trituration as described earlier (Szabo et al., 2002). Up to 8 testes were digested in 150 µl 0.25% trypsin (including 0.5 % BSA) at 36 °C for 15 min, tapping gently every 5 min. The trypsin reaction was stopped by adding 450 µl 20 % FBS in M2 medium (Cat. No. M7167 Sigma). The EGFP+ germ cells were purified by flow cytometry using a Beckman Coulter MoFlo Astrios sorter. ChIP-seq of fetal germ cells: ChIP was performed essentially according to Singh (Singh and Szabó, 2012). For the 100K ZBTB43 ChIP and 100K IgG ChIP, 100,000 FACS purified EGFP+ germ cells were pelleted down at 3000 rpm for 6 min at room temperature. Crosslinking was done by adding 500 µl of 1 % formaldehyde in 1 X PBS, followed by rotating on a platform gently at room temperature for 10 min. 25 µl of 1.25 M glycine was added into each sample to stop crosslinking. Germ cells were pelleted down at 728 rcf for 10 min at room temperature. The cell pellets were suspended in 100 µl of ChIP lysis buffer (Singh and Szabó, 2012) and were snap frozen in liquid nitrogen and kept at -80 °C till used for sonication. Before sonication, 20 µl of 6.5 X shearing buffer (65 mM Tris-HCl pH 8.0, 6.5 mM EDTA, 0.65 % SDS) was added to each sample. The sonication was carried out using a Covaris E220 Evolution ultrasound sonicator (PIP: 105; Duty Factor: 2; CPB: 200; Time: 12 min). For the 500K ZBTB43 ChIP sample and 500K IgG sample, crosslinking was done on combined batches of frozen prospermatogonia in 500 µl of 1 % formaldehyde in 1 X PBS. The reaction was stopped by adding 50 µl of 1.25 M Glycine. 100 µl of 6.5 X Shearing buffer was added into each sample. Sonication was carried out in 5 tubes. After sonication 1/10 of the sheared chromatin was taken out for ""input"". The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 µl/ml 50 X PIC. 1 µg of the anti-ZBTB43 antibody (Aviva Systems Biology, Cat. No. ARP39048-P050), or the control Rabbit-IgG (abcam Cat No: ab171870) was added per 100,000 cells and incubated overnight at 4 °C. The complex was washed, and the DNA was eluted as described earlier (Singh and Szabó, 2012). The captured DNA fraction was purified using phenol-chloroform extraction and ethanol precipitation and was stored at -80 °C until library preparation. Library preparation: The library was prepared with NEBNext® Ultra™ II DNA Library Prep Kit (New England Biolabs, Cat. No. E7645S) according to the manufacturer's protocol as described online (dx.doi.org/10.17504/protocols.io.wvgfe3w). Briefly, end preparation, adapter ligation and PCR amplification with index primers was performed using the captured DNA. The library was cleaned up before sequencing using 1 X Ampure beads twice to remove the excess adaptors. Deep sequencing: Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). 150 bp, paired end sequencing was performed on an Illumina NovaSeq sequencer using a 150 bp high-output sequencing kit (v2) (Illumina Inc., San Diego, CA, USA) to a minimum depth of 40 M reads/library. Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.