title: GSM7111232: Mouse testes, RAD51 ChIP-SSDS, control, rep2; Mus musculus; ChIP-Seq
library_construction_protocol: One testis from the control and a pair of testes from Fignl1 cKO mice (14-18 weeks) were dissected, the tunica albuginea was removed, and seminiferous tubules were transferred into 10 ml of 1% formaldehyde (Pierce, 28908) in PBS (Nakarai, 14249-24) and immediately homogenized in a Dounce homogenizer with 20 strokes of the tight-fit pestle. The homogenate was passed through a 70 m cell strainer (Falcon, 352350), and after a total of 10 min of incubation at room temperature, crosslinking was quenched by adding 2.5 M glycine to a final concentration of 125 mM. After 5 min of incubation at room temperature, cells were pelleted by centrifugation at 900 x g for 5 min at 4℃, washed once with 10 ml of cold PBS, and suspended in 1 ml of cold PBS. The cell suspension was transferred to a 1.5 ml tube, centrifuged at 900 x g for 5 min at 4℃, and after removal of the supernatant, the cell pellet was snap-frozen on liquid nitrogen and stored at -80℃. The frozen cell pellet was suspended in 500 μl of cell lysis buffer (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8.0) supplemented with protease inhibitor (Roche, 04693159001), incubated for 10 min on ice, centrifuged at 300 x g for 3 min at 4℃, and the nuclei pellet was resuspended in 500 l of lysis wash buffer (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8.0) supplemented with protease inhibitor. After centrifugation at 300 x g for 3 min at 4℃, the nuclei pellet was resuspended in 200 l of RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with protease inhibitor and split into two ~130 μl aliquots and sonicated on the Covaris M220 for 5 min 30 sec at 5-9oC with a setting of Peak Power 50.0, Duty Factor 20.0, and Cycles/Burst 200. Sheared nuclei were combined, and after adding 700 l of RIPA buffer, centrifuged at 15,000 rpm for 15 min at 4℃ to remove insoluble debris. 800 l of the chromatin-containing supernatant was mixed with 50 l of Dynabeads protein G bound to 9.7 g of RAD51 antibody (Novus, NB100-148) and incubated overnight on a rotating wheel at 4℃ for immunoprecipitation. Beads were washed once with 1 ml of cold ChIP wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml of cold ChIP wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml of cold ChIP wash buffer 3 (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and twice with 1 ml of cold TE (1 mM EDTA and 10 mM Tris-HCl pH 8.0), followed by elution with 100 l of elution buffer (0.1 M NaHCO3 and 1% SDS) for 30 min at 65℃ with vortexing every 10 min. The elutes were incubated overnight at 65℃ for de-crosslinking, neutralized by adding 4 l of 1M Tris-HCl (pH 6.5) and 2 μl of 0.5 M EDTA, and RNA was digested by adding 2 l of 10 mg/ml RNase-A and incubation for 15 min 37oC. After adding 5 l of 10 mg/ml Proteinase-K (Wako, 165-21043) and incubation for 1 h at 55℃, DNA was purified using QIAquick PCR Purification Kit (QIAGEN). 7-10 ng of ChIP DNA was further sonicated on the Covaris M220 for 8 min 30 sec at room temperature with a setting of Peak Power 75.0, Duty Factor 10.0, and Cycles/Burst 200, followed by end-repair and dA-tailing using ChIP-Seq Library Prep Reagent Set for Illumina (NEB, E6200L). DNA was incubated for 3 min at 95℃ and returned to room temperature for kinetic enrichment for single-stranded DNA. After adapter ligation using IDT for Illumina TruSeq DNA UD Indexes (Illumina, 20022370), SSDS libraries were amplified during 15 cycles of PCR using TruSeq DNA Nano LP (Illumina, 20016328) and purified with AMPureXP beads (Beckman Coulter, A63880).