GSM6276538: ChIP-seq with BORIS Abs in NIH3T3+BORIS cells 4; Mus musculus; ChIP-Seq
ChIP-seq protocol: 2x106 asynchronously growing cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 10 min, washed twice with 1×phosphate buffered saline (PBS), and resuspended in chromatin immunoprecipitation (ChIP) lysis buffer (150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 20 mM Tris–HCl pH8.0, 2 mM EDTA). Chromatin was sheared to 200–600 bp and immunoprecipitated with 5 μg of antibody. Immunoprecipitated DNA was purified with QIAquick columns (ZymaResearch). ATAC-seq protocol:NIH3T3 cells (5 × 104) were pelleted by centrifugation and resuspended in 50 µL of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), gently pipetting up and down 10 times. Nuclei were pelleted by centrifugation at 2500g for 10 min at 4°C and resuspended in 25 µL of 2x TD Buffer (Illumina Cat #FC-121-1030), containing 8 µL of Tn5 Transposes (Illumina Cat #FC-121-1030) and 17 µL of nuclease free water. The rection was incubated for 30 min at 37°C. Tagmented DNA was purified by using a Zymo Research DNA Clean & Concentrator™-5 - Capped Columns. RNA-seq protocol:The RNA sequencing library preparation and sequencing procedures were carried out according to Illumina protocols. Briefly, poly(A)-mRNA was purified from 5 μg of RNA with streptavidin-coated magnetic beads. After chemical fragmentation, mRNA fragments were reverse-transcribed and converted into double-stranded cDNA. Following end repair and A-tailing, paired-end adaptors were ligated to the ends of the DNA fragments. The ligated products were amplified with 18 cycles of PCR followed by purification using AMPure beads (Beckman Coulter). The enriched libraries were diluted to a final concentration of 5 nM. ChIP-seq libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA) or NEBNext UltraII DNA Library Prep Kit for Illumina (BioLabs, catalog # E7645S).  Libraries were amplified using 15 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq library were prepared using TruSeq RNA Library Prep Kit (Illumina, Inc., USA) ATAC-seq libraries were amplified using NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs). The libraries were size-selected  by adding 1.8X volume Agencourt AMPure XP beads (Beckman, 63881). CAGE-seq libraries were prepared by DNAFORM (Yokohama, Kanagawa, Japan).