GSM8772933: NIH3T3, Brf1 siRNA, mock-treated, CPSF30 (input); Mus musculus; ChIP-Seq
NIH3T3 mouse fibroblast cells plated in a 10-cm TC-treated dish were cross-linked in 1% formaldehyde in DPBS (Gibco) for 5 minutes at room temperature, quenched in 0.125 M glycine, and washed twice 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 5 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. 40 μg of chromatin was incubated with 10 μg rabbit polyclonal anti-POLR3A (Abcam ab96328), anti-CPSF30 (Proteintech 15023-1-AP), or rabbit IgG (Abcam ab37415) overnight. 25 μl of mixed protein A and G Dynabeads (Thermo Fisher) was 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 using 100 μl of 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) and eluted in 50 μl of nuclease-free water. DNA was used for prepping 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.