GSM8244162: SSDS-DMC1_Nbs1dB_rep2; Mus musculus; ChIP-Seq
ChIP SSDS was performed following the previously published (ref. 1, 2, 3). Fresh or frozen testes of wild-type or Nbs1∆B were placed in room temperature 1% PFA (16% stock, Pierce) in PBS and incubated for 15 min with gentle agitation. The crosslinking was stopped by adding 2.5 M glycine to a final concentration of 125 mM, followed by 5 min incubation at room temperature. Fixed cells were immediately homogenized in a Dounce homogenizer with 20 strokes of the tight-fitting pestle, then passed through a 70 μm cell strainer. The nuclei were prepared as described (ref. 2), resuspended in 900 μl shearing buffer (0.1% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with protease inhibitor (Roche), split into two ∼500 μl aliquots and sonicated to fragment sizes ranging 500–1000 bp with a Covaris system (E220 Focused-ultrasonicator, microtube-500) using the following parameters: delay 300 s then three cycles of [peak power 75 W, duty factor 10%, cycles/burst 200, duration 150 s, and delay 60 s]. Sheared nuclei were centrifuged at 12,000 g for 10 min at 4 °C to remove debris. The sonicated chromatin was then transferred to 3 ml Slide-A-Lyzer G2 dialysis cassette (Pierce) and dialyzed with ChIP buffer (16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) for 5 hr at 4 °C with constant but slow stirring. For each immunoprecipitation, 20 μl of anti-DMC1 (Abcam, ab11054), 10 μl of anti-RAD51 (Novus Biologicals, NB100-148), or 10 μl of anti-RPA2 (Abcam, ab76420) was added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation.  As described previously (ref. 1, 2, 3), protein G or A Dynabeads were added to chromatin and antibody mixture and incubated 2 hr at 4 °C, then immunoprecipitated DNA was washed and eluted.  (ref. 1) Khil, et al., Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA, Genome Res. May; 22(5): 957–965. (2012).  (ref. 2) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018).  (ref. 3) Hinch, et al., The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates. Mol Cell 79(4): 689-701 e610. (2020) To reverse DNA-protein crosslinks, 12 μl of 5 M NaCl was added to 300 μl of eluted sample and incubated overnight at 65 °C. DNA was cleaned with MinElute PCR Cleanup Kit (Qiagen) after proteinase K (Thermo) treatment. Following end repair and dA-tailing, DNA was incubated at 95 °C for 3 min and cooled to room temperature to enrich for single-stranded DNA as described before (ref. 1). After ligation with sequencing adaptors, SSDS library was amplified with 12 cycles of PCR.   After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. We obtained paired-end reads of 100 bp. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NovaSeq 6000.  (ref. 1) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018).