GSM5964291: NS131-DLD1-DMSO-D7-C; Homo sapiens; RNA-Seq
RNA was isolated using Trizol reagent (ThermoFisher) or the AllPrep DNA/RNA/protein mini kit (Qiagen #80004) according to manufacturer recommendations. For ChIP-seq, HCT116, HCT8, DLD1, and SW620 cells were cross-linked with formaldehyde (1% final) for 10 min at room temperature. Cross-linked cells were quenched with glycine (125 mM final) for 5 min, followed by two washes in cold PBS. Nuclei were then isolated from 20 million cells as previously described (67), and chromatin was sheared to 250-bp average size using a Covaris S220. Immunoprecipitations were performed using 1000 μg of sheared chromatin lysate and 5 μg of p53 antibody (p53 D07; Santa Cruz, sc-47698) preconjugated to protein G beads (Invitrogen). ChIP reactions were incubated for 16 hours at 4°C with rotation and then washed four times in wash buffer [50 mM Hepes-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, and 0.5% N-laurylsarcosine], followed by one wash in ChIP final wash buffer (1× tris-EDTA (TE) Buffer and 50 mM NaCl). Immunoprecipitated DNA was eluted from washed beads, reverse cross-linked overnight, purified, and used to construct libraries. Clontech/Takara SMARTer Stranded Total RNA-seq PICO v2 kit (Clontech/Takara 634414) for RNA-seq. For ChIP-seq, libraries were prepared using NEBNext Ultra reagents (New England Biolabs).