GSM5530935: LNCap cells_siGATA2_V_SOX9_rep1; Homo sapiens; ChIP-Seq
For ChIP-seq, cells were crosslinked with 1% formaldehyde for 10 min on horizontal rotator at room temperature and cell pellets were collected and subjected to sonication. The sheared chromatin was then diluted and immunoprecipitated with 4 µg of specific antibodies at 4°C overnight. After washing, chromatin complexes were eluted with elution buffer and crosslinking was reversed at 65°C overnight. DNA fragments were purified with the QIAquick PCR purification kit. For ATAC-seq, 50,000 cells were resuspended in cold ATAC-seq resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2). Cell nuclei were then prepared by incubation in 50 μl of ATAC-seq resuspendsion buffer containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin on ice for 3 min. After centrifugation, nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer ,2.5 μl Nextera Tn5 transposase (Illuminar), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water), and incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 rpm. Transposed fragments were then purified with a Zymo DNA Clean and Concentrator-5 Kit. All libraries showed sufficient amplification after the 5 pre-amplification cycles and quantified using the KAPA Library Quantification Kit. For ChIP-seq, cells were crosslinked with 1% formaldehyde for 10 min on horizontal rotator at room temperature and cell pellets were collected and subjected to sonication. The sheared chromatin was then diluted and immunoprecipitated with 4 µg of specific antibodies at 4°C overnight. After washing, chromatin complexes were eluted with elution buffer and crosslinking was reversed at 65°C overnight. DNA fragments were purified with the QIAquick PCR purification kit. For ATAC-seq, the library preparation was performed with Nextera DNA Library Prep Kit for Illumina following the manufacturer's instructions. The cDNA molecules were amplified by 8 cycles of PCR. Non-size selection libraries were then sequenced for using Illumina Novaseq 6000 at the Duke sequencing core. For ChIP-seq, the eluted ChIP DNA was used for library preparation with NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina according to the manufacturer's protocol. The library was ampliﬁed with 12 PCR cycles and prepared with gel-based size selection.