GSM9277841: no.tag-dsae2-Rep2; Saccharomyces cerevisiae; ChIP-Seq
For each meiotic time point, 2 x 108 cells were fixed in paraformaldehyde (1% final). FLAG-Tel1 cells were fixed for 30min, while FLAG-Spo11 strain were fixed for 15min, quenched by glycine for 5min (125mM final) and washed in TBS 1X two times. Cells were lysed using mechanical lysis (Fastprep, 4 cycles at 6.5M/sec 30sec) together with a Lysis buffer (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA pH8, 1% Triton X100, 0.1% Sodium Deoxycholate) complemented with protease inhibitor cocktail tablet (Roche, Ref 05 892 791 001), PMSF (1mM final), pepstatin (0.1mM final) and leupeptin (1mM final). After cell lysis, chromatin was sheared by sonication into 500 – 1000pb fragment (30sec ON/30sec OFF, 6 cycles, 4°C, Diagenode Bioruptor Pico). 10µl was kept aside as the input, and samples were divided in two halves, for minus and plus antibody treatment. FLAG-Tel1 immunoprecipitation was performed with 2µl of anti-FLAG antibody (M2 monoclonal, Sigma, Ref F1804) and incubation at 4°C for 2 hours. Pan-IgG mouse magnetic beads were incorporated for 2 more hours at 4°C. IP samples were washed by a 500mM NaCl containing lysis buffer and LiCl buffer (10mM Tris-HCl pH8, 0.25LiCl, 1mM EDTA pH8, 0.5% Triton X-100, 0.5% Sodium Deoxycholate). Chromatin was eluted from magnetic beads in elution buffer (TE 1X, 0.5% SDS) at 65°C for 10min. DNA-protein interaction was disrupted by incubation at 65°C overnight for IP and input samples. RNA was digested by RNAse A treatment (20µg total, 15min at 37°C) and proteins by proteinase K treatment (100µg total, 1hour at 42°C). DNA was purified on column (Qiagen DNA purification kit). Tel1 and Spo11 ChIP-seq were performed following the ChIP-qPCR protocol with some modifications. For both FLAG-Tel1 and FLAG-Spo11 strain, cells were fixed in paraformaldehyde for 30min. Protein G magnetic beads were washed (0.1M NaHPO4 pH7.5, 0.01%Tween20, pH8.2) and saturated with BSA (0.1M NaP, 0.01% Tween20, 5mg/ml BSA) for 3hours at 4°C. Anti-FLAG antibody (M2 Monoclonal, Sigma) was pre-incubated with saturated-magnetic beads for 4hours at 4°C. Cell lysis was performed as for the ChIP-qPCR protocol. Chromatin shearing was performed in Diagenode tube 15ml with beads (900 – 1000mg) by 7 cycles of sonication (30sec ON/30sec OFF, 4°C). Chromatin was immunoprecipitated with antibody-protein G mix by overnight incubation at 4°C. Samples were washed as for the ChIP-seq protocol, and DNA was eluted with IPure v2 Diagenode kit. DNA quantity was measured by QuBit and fragmentation quality was controlled by Agilent chip (Bio-analyser). DNA was sequenced with a NovaSeq sequencer (Illumina) using a 100nt paired-end protocol (TruSeq) at the Institut Curie sequencing facility (Paris, France).