GSM9110586: Jurkat, TRAIL-treated, replicate 1, V5 ChIP; Homo sapiens; ChIP-Seq
Cells were then cross-linked in 1% formaldehyde in DPBS (Gibco) for 5 minutes at room temperature, quenched in 0.125 M glycine, and washed three times with DPBS (Gibco). Cross-linked cells were harvested and lysed with 1 ml ChIP lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) by rotation for 10 min. Nuclei were collected by centrifugation at 1,700 × g for 5 min at 4°C, washed once with ChIP shearing buffer (50 mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.1% SDS), and then resuspended in 1 ml of ChIP shearing buffer. Chromatin was then sheared for 8 min using a Covaris S220 focused ultrasonicator at 140 power, a 5% duty cycle, and 200 bursts/cycle. Chromatin was spun at 15,000 × g for 5 min at 4°C, and the pellet was discarded. 10 μg of chromatin was incubated with 5 μg rabbit monoclonal anti-RPB1 NTD (clone D8L4Y; Cell Signaling 14958), rabbit polyclonal anti-V5 (Abcam ab15828), or rabbit IgG (Abcam ab37415) overnight. 6xHis pulldown was attempted in parallel but yielded insufficient quality and was excluded from further analysis. 25 μl of mixed protein A and G Dynabeads (Thermo Fisher) were added, and the tubes were rotated for 2 hours at 4°C. Dynabeads were then washed with low-salt immune complex (20 mM Tris pH 8.0, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 0.1% SDS), high-salt immune complex (20 mM Tris pH 8.0, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 0.1% SDS), lithium chloride immune complex (10 mM Tris pH 8.0, 0.25 M LiCl, 1% NP-40, 1% deoxycholic acid, 1 mM EDTA), and Tris-EDTA for 5 min each at 4°C with rotation. DNA was eluted from the beads in elution buffer (150 mM NaCl, 50 μg/ml proteinase K) and incubated at 50°C for 2 h and then at 65°C overnight. DNA was purified using an Oligo Clean and Concentrator kit (Zymo), eluted in nuclease-free water, and then used to generate libraries for ChIP-seq. Libraries for ChIP-seq were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1). Equal amounts of sample eluted DNA and spike-in DNA (yeast spike-in DNA – Cell Signaling) were used within each experiment and amplified for 3-10 cycles based on NEB recommendations and sequenced on a NovaSeq X 10B (Illumina) with 150 paired-end reads.