GSM6757956: 6_FUS2 H3K27Ac; Mus musculus; ChIP-Seq
Sorted NeuN+ nuclei were  subjected to ChIP-sequencing using antibodies against the 3 histone marks: H3K27ac, H3K4me3 and H4K12ac. They were resuspended in Nuclei lysis buffer (10 mM EDTA, 50mM Tris-HCL pH8.1, 1mM NaBu and 1% SDS in ddH2O) with PIC in a 15 ml TPX tube. Nuclei were sonicated using the sonicator (Bioruptor Plus sonication device, Diagenode) with 17 cycle of 30sec ON / 30sec OFF on High Power with breaks to vortex tubes every 5 min. 4uL of supernatants were checked on 1,5% agarose gel to confirm DNA fragmentation of approximately bellow 500bp after sonication and between 500bp and 100bp after a rapid decrosslinking protocol. Sonicated chromatin was centrifuged at 4°C, 14000g for 10 min to get rid of debris. Supernatants were diluted in ChIP Dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl in ddH2O) to obtain a final concentration of 0,1%SDS. Approximately 40 000 nuclei were saved before IP for total input chromatin and the rest of the sample were split in a minimum of 400 000 nuclei per ChIP. Samples were incubated overnight (O/N) at 4°C with the following primary Ab: rabbit anti-H4K12ac, 9ug/ChIP (Diagenode, Ref: C15410331), rabbit anti-H3K4me3, 4,25ug/ChIP (Diagenode, Ref: C15410003-10), rabbit anti-H3K27ac, 3,1ug/ChIP (Abcam, Ref: ab4729). 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 μl 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 10 ng 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 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.