GSM9568537: U2OS_RFP_EGF_Input_MYH9; Homo sapiens; ChIP-Seq
Cells were fixed in 1% formaldehyde for 5 minutes. After quenching and cell lysis, chromatin was sonicated using a FB120 sonicator with CL-18 tip (Fisher Scientific), Libraries were prepared according to Takara Bio USA, Inc. DNA SMART™ ChIP Seq Kit User Manual (Part# 634866 and 634867). To generate ChIP-seq libraries with the DNA SMART ChIP-Seq Kit, a priming site is first added to the 3' end of the DNA template using the Terminal Deoxynucleotidyl Transferase. This is followed by annealing of a proprietary DNA SMART Poly(dA) Primer, which anneals to the T-tail added by the Terminal Deoxynucleotidyl Transferase. This primer is then used by the SMARTScribe™ Reverse Transcriptase (RT) to copy the DNA strand. When the SMARTScribe RT reaches the 5' end of the DNA template, the enzyme's terminal transferase activity adds a few additional nucleotides to the 3' end of the newly synthesized DNA. The carefully designed DNA SMART Oligonucleotide base-pairs with these additional non-template nucleotides and creates an extended template, enabling the SMARTScribe RT to continue replicating to the end of the oligonucleotide. Sequencing libraries are then generated by PCR-mediated addition of Illumina adapters using primers compatible with regions on the DNA SMART Poly(dA) Primer and the DNA SMART Oligonucleotide. 1ng of DNA was used as starting material for input and IP samples. Libraries were amplified using 15 cycles on the thermocycler. Post amplification libraries were double size selected with 200-500bp in length using AMPure XP beads from Beckman Coulter. The quality control of the libraries was performed on an Agilent Technologies 4150 TapeStation System using an Agilent high sensitivity D5000 chip. Multiplex DNA libraries are normalized to 10nM and then pooled in equal volumes with each unique barcoded library. The purified DNA was captured on an Illumina flow cell for cluster generation.