title: GSM1289334: MCF7_PMA_Total_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP samples were prepared with anti-PKC-theta (sc-212, SantaCruz), according to the protocol supplied by Upstate Biotechnology. 10ng of PKC-theta immunoprecipitated ChIP-DNA, and the corresponding total input (TI), were used to prepare the ChIP-seq DNA library using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina® (New England BioLabs Inc, NEB#E6240L) according to the manufacturer’s instructions. ChIP-DNA size was selected with AMPure XP beads (Beckman Coulter, Inc., Part #: A63881). The selected DNA was repaired with NEBNext End Repair Enzyme Mix and Buffer, followed by treatment with Klenow fragment (3’à5’ exo-) to generate “A” base overhangs. Subsequent adaptor ligation of the dA-tailed DNA was performed with 1.5μM NEBNext adaptor primers, Quick Ligation Reaction Buffer, and Quick T4 DNA ligase. AMPure beads were used to isolate library fragments ranging between 175 and 225bp in size and the purified DNA was combined with the NEBNext High-Fidelity 2X PCR Master mix, 25μM Universal PCR primer, and 25μM index primer for multiplexing purposes (New England BioLabs Inc, NEBNext® Multiplex Oligos for Illumina®, NEB#E7335L). A total of 15 cycles were used for PCR amplification of the adaptor-ligated DNA. The resultant ChIP-DNA library was subjected to DNA purification with AMPure beads. The quality of the ChIP-DNA library was assessed on a Bioanalyzer. A total of 2nM was captured on the Illumina flow cell for cluster generation
attributes.tag: GEO Accession
attributes.value: GSM1289334