title: ZBTB43 eliminates Z-DNA in mouse prospermatogonia
design: MIRA-seq MIRA-seq was carried out according to Jung (Jung et al., 2015). GST-MBD2b and His-MBD3L1 preparation: GST-MBD2b and His-MBD3L1 was purified from E. coli against GST, and His tags respectively. Both proteins were stored in salt-dialysis buffer (50 mM HEPES pH 7.4, 50 % glycerol, 5 mM 2-mercaptoethanol, and 150 mM NaCl) at -20 °C for no more than 6 months. Sperm collection by swim-up and DNA preparation: adult male mice were sacrificed at 3 months of age by CO2 asphyxiation and pairs of cauda epididymis were dissected and cleaned from fat tissue in 1 X PBS under microscope. Caudae were placed into 1 ml of M16 buffer, nicked with a blade three times, and were transferred with the buffer into a round bottom tube. 4 ml of additional M16 buffer was gently layered over the caudae. The samples were incubated, lid open, in a cell culture incubator at 37 °C with 5 % CO2 for 2 h. The top 4 ml was transferred into 15 ml tube without touching the bottom and the cell number was counted. The motile spermatozoa were pelleted down at 3000 RPM for 6 min. The sperm pellet was then suspended in 1ml sperm lysis buffer (0.1 M DTT and 2 mg/ml Proteinase K in 10 mM Tris-HCL pH 8.0, 10 mM EDTA and 1 % SDS) per cauda and was incubated at 55 °C overnight. The DNA was isolated 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. MIRA reaction: MIRA proteins, 1 µg of GST-MBD2b and 1 µg His-MBD3L1, and the MagneGST beads (REF V8611, Promega) were preblocked with 500 ng unmethylated E.Coli genomic DNA in 1 X MIRA binding buffer (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 1 mM DTT, 10 mM MgCl2, and 0.1 % Triton-X-100) at 4 °C for 20 min. 500 ng fragmented sperm DNA was combined with the with preblocked MBD2b and MBD3L1 proteins and the binding reaction was carried out at 4 °C, overnight with rotation. Next day, 5 µl preblocked MagneGST beads (REF V8611, Promega) were added to each MIRA rection and the bound fraction was captured by incubating at 4 °C for 1 h with rotation. The beads were collected using a magnetic stand. They were washed three times using 900 µl prechilled MIRA wash buffer (10 mM Tris-HCl pH 7.5, 400 mM NaCl, and 0.1 % Triton-X-100). After washing, the beads were captured using a magnetic stand and DNA was collected by adding 500 µl buffer PB from the Qiagen PCR purification kit (Catalog number: 28105) and DNA was isolated according to the kit's instructions. MIRA library: Libraries were prepared by the Van Andel Genomics Core form 2.5 ng of input material using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems, Wilmington, MA USA). Prior to PCR amplification, end repaired and A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA). 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). 75 bp, single end sequencing was performed on an Illumina NextSeq 500 sequencer using a 75 bp mid-output sequencing kit (v2) (Illumina Inc., San Diego, CA, USA). 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.