title: GSM2561940: SDS1 Set8KO WT PolII replicate 1; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-seq, extracts from crosslinked liver tissues were immunoprecipitated by antibodies against RNA Pol-II (Euromedex IPB-7G5), Rabbit polyclonal anti-phospho Ser2-RNA Pol-II CTD (Abcam ab5095), Rabbit polyclonal anti-PHF8 (Abcam ab36068), H4K20Me1 (Abcam ab9051) as described in Sarris et al., 2016, Cancer Cell Vol: 14. pp 354-366). About 10 ng of the immunoprecipitated DNA and input DNA was used for library preparation following the optimized workflow of Ion Proton system (Life Technologies). Briefly, ChIPseq DNA libraries were created using the Ion Plus Fragment Library Kit (ThermoFisher Scientific) protocol with the following modifications: approximately 2ng of sheared chromatin immunoprecipitated DNA was diluted with ddH2O in a final volume of 79μl. For DNA samples with undetected concentration the whole eluted volume was used (~25μl) and added to the required amount of ddH2O. DNA was End-repaired by the addition of 20μl of End-repair buffer and 1μl enzyme per sample followed by 30min incubation at RT. Samples were purified with Agencourt AMPure XP beads (Beckman Coulter) at a ratio of 1.8 and, after subsequent washes with 70%EtOH, were eluted in 40μl Low TE. Adaptors were then ligated to the DNA in the presence of 10μl ligase buffer and 2μl ligase enzyme. 1μl of universal IonXpress P1 adaptor was added in all samples with 1μl of barcoded IonXpress adaptor, 1-96. The reaction was diluted in ddH2O to a final volume of 100μl and incubated for 30min at RT. After a further purification with Agencourt AMPure XP beads at a ratio of 1.5, the  samples, eluted in 25μl of LowTE, were amplified using 100ul Platinum PCR Supermix High Fidelity and 5μl Library amplification primer mix. Each sample was divided into two 65μl reactions and amplified using the following thermal cycling protocol: 72°C-20’/95°C-5’/(97°C-15’’,60°C-15’’,70°C-1’)*17cycles/70°C-5’. The 2 reactions for each sample were mixed again and the samples were purified using a 2 step purification protocol. During the first step, Agencourt AMPure XP beads to sample ratio of 0.8 was used, to remove fragments of 400bp or more. The supernatant was used for a 2nd purification using Agencourt AMPure XP beads ratio of 0.5 and eluted in in 25μl ddH2O. The ChIP DNA libraries with a mean fragment length of 200bp were quantified on an Agilent bioanalyzer and 40pM of each library was templated using the Ion Chef instrument, loaded on a Ion P1 V3 chip and sequenced on an Ion Proton sequencer.
attributes.tag: GEO Accession
attributes.value: GSM2561940