8WG16_ChIP-seq_siFus_siEwsr1_siTaf15
N2A cells were grown in MEM+10% FCS.Cells were transfected with siRNAs against Fus, Ewsr1 and Taf15 using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer?s instructions. ChIP experiments were performed 48hrs after transfection.
N2A cells in a 15-cm dish were subjected to crosslinking in 1% formaldehyde for 10 min at RT, then quenched with 125 mM glycine for 5 min. Cells were washed, and the cell pellets were suspended in 1 ml lysis buffer. The lysates were  sheared to yield fragments of 100-300 bp. Samples were centrifuged to remove insoluble material, and the supernatant containing DNA-protein complexes was collected. 8WG16 Ab (2 ?g) and 20 ?l of Dynabeads proteinG were added to the chromatin lysate, and were incubated on a rocking platform overnight at 4 ?C. Immune-complexes were washed extemsively. Immune-complexes were eluted with elution buffer . The samples were added with 10 ?l of 5 M NaCl and 5 ?l of 5 mg/ml proteinase K, and incubated at 65 ?C for 4 hrs to reverse the DNA-protein crosslinks. The final DNA fragments were purified with QIAquick PCR Purification Kit (Qiagen). ChIP-DNAs were gel-purified and transformed into libraries using  the NEB DNA library preparation kit for Illumina (cat # E6000L) according to the  manufacturer's instructions, and sequenced on Hiseq2000 DNA sequencing instrument at Otogenetics Corp. Total input DNA was also sequenced.