title: GSM8241548: MRE11-ChIP_wt_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: MRE11-ChIP was performed following the previously published DISCOVER-seq protocol (ref. 1) Fresh testes of 7 weeks old wild-type, Atm–/–, or Mre11-cHN mice were dissociated into single cells as described before (ref. 2) and resuspended in DMEM. Testis cells from mice at indicated age and genotype were obtained as described previously (ref. 1). PFA (16% stock, Pierce) was added to a final concentration of 1% and incubated for 15 min with gentle agitation, then the crosslinking was stopped by adding 2.5 M glycine to a final concentration of 125 mM, followed by 3 min incubation on ice. The nuclei were prepared as described (ref. 1) and up to 10 million nuclei were resuspended in 500 μl lysis buffer (0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) supplemented with protease inhibitor (Roche), and sonicated to fragment sizes ranging 200–300 bp with a Covaris system (E220 Focused-ultrasonicator, microtube-500) using the following parameters: delay 300 s then six cycles of [peak power 75 W, duty factor 10%, cycles/burst 200, duration 150 s, and delay 60 s]. Sheared nuclei were centrifuged at 20,000 g for 10 min at 4 °C to remove debris and Triton-X (1% (vol/vol) final concentration) and lysis buffer were added to make it a total volume of 3 ml. (ref. 1) Wienert, et al., CRISPR off-target detection with DISCOVER-seq, Nat Protoc 15(5): 1775-1799 (2020). (ref. 2) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020). For each immunoprecipitation, 10 μl of anti-MRE11 (Novus Biologicals NB100-142) was pre-mixed with 100 μl protein A Dynabeads, then added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation. Immunoprecipitated DNA was eluted and crosslinks were reversed as described (ref. 1). ChIP sequencing libraries were prepared from cleaned DNA as described for SSDS libraries (ref. 2) without the boiling step after the end repair and dA-tailing steps. After ligation with sequencing adaptors, the library was amplified by 15 cycles of PCR. The amplified library was purified.  Reads for S1, Exo7/T, or ExoT-seq were counted at which a nucleotide next to biotinylated adaptor DNA was mapped (this corresponds to a position of ssDNA). Maps were analyzed using R version 4.0.3 <http://www.r-project.org>.   (ref. 1) Wienert, et al., CRISPR off-target detection with DISCOVER-seq, Nat Protoc 15(5): 1775-1799 (2020). (ref. 2) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018). (ref. 3) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).