GSM9350472: ASCL1 ChIP-seq on CORL47 cells expressing empty vector Rep2; Homo sapiens; ChIP-Seq
•Cells were collected and then cross-linked by incubation with 2 mM disuccinimidyl glutarate (DSG) prior to fixation step. Then cells were fixed in 1% paraformaldehyde. Crosslinking was then quenched by adding freshly made 1.25 M glycine. The cells were resuspended in 1 mL of lysis buffer (50 mM Hepes-NaOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100 supplemented with a protease inhibitors and phosphatase inhibitors) to collect nuclei. The intact nuclei were transferred to a 1 mL AFA fiber tube (Covaris #520130). The chromatin was then sonicated using a Covaris E220 sonicator. For each sample, 5% of the total lysate was used for the ChIP-seq input DNA and remaining lysate (95%) was used for either FLAG-ChIP or ASCL1-ChIP. 10 μg and 2 ug of FLAG and ASCL1 antibody, respectively, was  used for each ChIP. All samples were incubated overnight by rotating at 4°C. The next day all samples were washed through low salt, high salt and LiCl wash buffer. RNase A (Qiagen #19101) was then added to a final concentration of 0.2 mg/mL and the samples were incubated for 30 minutes at 37°C. To reverse the formaldehyde crosslinking and remove protein, proteinase K was added to a final concentration of 0.8 mg/mL and the samples were incubated for 30 minutes at 42°C and after that an additional 6 hours of incubation at 65°C. DNA was then collected and purified by QIAquick gel extraction kit (Qiagen #28706). DNA concentration was determined using the Nanodrop 8000 (Thermofisher Scientific) and samples were stored at −80°C. For all experiments, an isotype matched control IgG ChIP (CST, #3900) used at same total amount as the experimental ChIP antibody described above and was performed with an equal number of cells for each ChIP sample. ChIP-seq library prep and sequencing were performed by the Molecular Biology Core Facilities at Dana-Farber Cancer Institute. ChIPseq libraries were prepared using IDT XGen DNAseq library prep reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on an Illumina NovaSeq X Plus targeting 40 million 150bp read pairs.