title: ZBTB43 eliminates Z-DNA in mouse prospermatogonia
design: DNA-Protein affinity sequencing assay Affinity capture: His-MBP-ZBTB43 recombinant protein was purified from E.coli against His tag and stored in dialysis buffer (50 mM HEPES pH 7.4, 50 % glycerol, 5 mM 2-mercaptoethanol) at -80 °C. Unmethylated genomic DNA was purified from mouse TKO ES cells (Dnmt1-/- Dnmt3a-/- Dnmt3b-/-) ES (clone19) RBRC No.: AES0146 (Tsumura et al., 2006) with phenol-chloroform extraction and ethanol precipitation, and was sonicated to an average fragment size of 200 bp using a Covaris E220 Evolution ultrasound sonicator. Fully methylated DNA was prepared using M.SssI (NEB, Cat. No. M0226M) in fragmented mouse lung genomic DNA. Sperm DNA was prepared from WT and ZBTB43 mutant adulut mice. His-MBP-ZBTB43 protein (His-MBP as background control) was preincubated with MBP-magnetic beads (NEB, Cat. No. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the preincubated beads in a 200 µl reaction volume of 1 X binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl2, 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl2) at 37 °C for 2 h. After incubation, the beads were washed with 1 X binding buffer 3 times to remove the unbound DNA. The bound DNA was eluted in 1 X elution buffer (10 mM Tris-HCl pH 8.0, 10mM EDTA, 500 mM NaCl, % SDS, 10 mM maltose), and was collected by ethanol precipitation. The precipitated DNA was stored in -80 °C before library preparation. Library preparation: the library was prepared with NEBNext® Ultra™ II DNA Library Prep Kit (NEB Cat. No. E7645S) according to the manufacturer's instructions 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 and data analysis: Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.) and Promega QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S1, 100 bp sequencing kit (Illumina Inc., San Diego, CA, USA) to a minimum depth of 30 M reads/library. Base calling was done by Illumina RTA3 and output of NextSeq Control Software (NCS) v2.0 was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.DNA-Protein affinity sequencing assay