title: GSM3061161: ChIPseq_serum_H3K27me3_siTIP5; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP analysis was performed as previously described (Leone et al., 2017). Briefly, 1% formaldehyde was added to cultured cells to cross-link proteins to DNA. For histone ChIPs, isolated nuclei were then lysed and sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and pre-cleared with 10 μl packed Sepharose beads for 2 h at 4°C. Pre-cleared chromatin was incubated overnight with the indicated antibodies. The next day, Dynabeads protein-A (or -G, Millipore) were added and incubated for 4 h at 4°C. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform and ethanol precipitated. For TIP5 ChIPs, isolated and crosslinked nuclei were MNase digested in 400µl MNase digestion buffer (0.3M Sucrose, 50mM Tris pH 7.5, 30mM KCl, 7.5mM NaCl, 4mM MgCl2, 1mM CaCl2, 0.125% NP-40, 0.25% NaDeoxycholate, 2mM Na3VO4 supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche)) with 100U MNase (Roche) at 37°C for 1h. The digestion was then stopped with 5mM EDTA and the digested chromatin was solubilized in 1% SDS and three pulses of 30sec sonication using a Bioruptor ultrasonic cell disruptor (Diagenode). 200µg of pre-cleared chromatin was immunopurified with incubation of 30µl of Anti-FLAG M2 Affinity Gel (Sigma) or 30µl Anti-HA magnetic beads (Pierce) over night. The samples were subsequently washed, eluted and the DNA was purified as for histone ChIPs. For ChIPseq analyses, the quantity and quality of the isolated DNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) was used in the sfollowing steps. Briefly, ChIP samples (1 ng) was end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).
attributes.tag: GEO Accession
attributes.value: GSM3061161