title: NextSeq 550 sequencing; Myc binding to target sites in S2 cells (ChIPseq) in response to knockdown of Myc or the PAF1 complex component Atu/Leo1
design: Myc binding to target sites in S2 cells (ChIPseq) in response to knockdown of Myc or the PAF1 complex component Atu/Leo1
library_construction_protocol: S2 cells were left untreated or incubated with dsRNA against Atu or Myc. The cells were harvested 48h later. (HA-Leo1 expression was induced by addition of 125 M CuSo4. Cells were harvested 24h later)  Cells were cross-linked with 1% formaldehyde at 37C for 10 min and the reaction was stopped with 50 mM glycine. Cells were lysed and nuclei were resuspended in RIPA buffer. Sonication with a Branson sonifier was carried out until the majority of fragments showed nucleosomal size. Chromatin was immunoprecipitated with Myc antibody (rabbit -Myc) or control IgG from rabbit (Sigma) which were coupled to Protein A/G-Dynabeads (Invitrogen). DNA was purified with Phenol- Chloroform after elution of the bound chromatin with 1% SDS and reversion of the crosslink. Sequencing libraries were prepared with the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB #6240S/L) following the instruction manual. The cleanup steps were carried out using MinElute PCR Purification Kit (Qiagen) followed by gel electrophoresis for size selection.
attributes.tag: Experimental Factor: RNA interference
attributes.value: none
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: Myc