GSM6757980: WT1; Mus musculus; ChIP-Seq
The nuclei obtained from total tissue were resuspended in 700μL Nuclei lysis buffer containing only 0.2% SDS. Fixed nuclei were sonicated with 15 cycles of 30sec ON / 30sec OFF. We used a primary rabbit antibody anti-FUS 15μg/ChIP (Bethyl, Ref: A300-293A). On the next day samples were incubated with 50ul/ChIP magnetic beads (Diagenode, Dynabeads protein A, Ref: 10002D) at 4°C during 2h. Beads were previously washed three times in ChIP dilution buffer with 0,1% SDS and blocked O/N at 4°C with BSA. ChIP-DNA were then washed 5 min in several buffers, using a magnetic rack: Low salt, High Salt, LiCL and TE buffer. ChIP-DNA was then incubated in 300 ul elution buffer (1% SDS, 0.1 M NaHCO3 in ddH2O) at RT for 5 min and at 65°C for 10 min. Crosslinkings were reversed by addition of 0,2M NaCL, O/N at 65°C. Then DNAs were purified with RNAse (Abcam, Ref: ab52579) at 37°C for 30 min, and in Proteinase K buffer (Invitrogen, Ref: 100005393) at 45°C for 1h. Finally, DNAs were extracted with the MicroChIP Diapure columns (Diagenode, Ref: C03040001) and quantification of DNA concentrations were calculated using the Qubit. All samples reached the minimum of 2ug required for sequencing. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 2.5 to 10ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 + 4 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.