ZBTB43 eliminates Z-DNA in mouse prospermatogonia
Purification of fetal male germ cells of the testis: To obtain fetuses for germ cell purification, OG2 (Szabo et al., 2002) females (Oct4-EGFPTg/Tg; Zbtb43+/+) were crossed with mutant (Zbtb43-/-; Oct4-EGFPTg/0) or wild type males. Testicles were dissected from the resulting male fetuses at different days of gestation, at 15.5 days post coitum (dpc) and were dissociated to single cell suspension using trypsin digestion and trituration as described earlier 16. 2-10 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 (Sigma, M7167). The EGFP+ germ cells were purified by flow cytometry using a Beckman Coulter MoFlo Astrios sorter. Cut&Run: Cut&Run was performed according to Skene (Skene et al., 2017) Briefly, the FACS-sorted germ cells and somatic cells were pelleted down at 3000 rpm for 6 min at room temperature, and were washed 3 times using 1 ml R/T wash buffer. For less than 5000 cells, washing was done only once. 10 µl of proA-coated beads was added into each sample in 1ml R/T wash buffer, followed by rotating for 10 min at room temperature. The anti-ZBTB43 antibody (Aviva Systems Biology, Cat. No. ARP39048-P050), or the control IgG (Rabbit-IgG: ab171870, abcam) was added at 1:100 dilution, and the anti-Z-DNA antibody, Z22 (Absoluteantibody, Cat. No Ab00783-3.0) was added at 1:200 dilution in 100 µl 5% DIG-antibody buffer, and the sample was incubated overnight at 4°C. After incubation, the complex was washed 2 times with DIG-wash buffer. The pAG-MNase (EpiCypher, Cat. No. 15-1016,) was added at 1:25 dilution in 50 µl DIG-wash buffer, followed by incubation at 4 °C for 1 h, with tapping every 5 min to suspend the complex. The captured DNA fraction was purified using phenol-chloroform extraction and ethanol precipitation and was stored at -80 °C until library preparation. It was necessary to pool several captured DNA aliquots to equalize cell numbers to 50,000 or 100,000 in the 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 0.9 X Ampure beads 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 NextSeq 500 sequencer using a 150 bp high-output sequencing kit (v2) (Illumina Inc., San Diego, CA, USA) to a minimum depth of 20 M-30 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.