GSM4868062: Mock 2 CLIP; Homo sapiens; RIP-Seq
Whole-cell lysates were generated in CLIP lysis buffer (50 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40, 0.2% Triton X-100, 0.5% N-lauroylsarcosine) and briefly sonicated using a probe-tip Branson sonicator to solubilize chromatin. Eluted cDNA was transferred to a new PCR tube containing 15 mL of 2X Phusion HF-PCR Master Mix (NEB), 0.5 mL of 30 mM P3/P6 PCR1 oligo mix and 0.5 ml of 15x SYBR Green I (ThermoFisher Scientific). Real-time quantitative PCR was preformed: 98°C 2 min, 15 cycles of 98°C 15 seconds, 65°C 30 seconds, 72°C, 30 seconds, with data acquisition set to the 72°C extension. PCR1 reactions were cleaned up by adding of 4.5 mL of isopropanol, 54 mL of AMPure XP beads and incubation for 10 min. Beads were washed once with 80% ethanol, dried for 5 min, and eluted in 15 ml of water. Illumina flow cell adaptors were added by adding 15 mL 2X Phusion HF-PCR Master Mix and 0.4 mL P3solexa/P6solexa oligo mix and amplified:  98°C 2 min, 3 cycles of 98°C 15 seconds, 65°C 30 seconds, 72°C, 30s seconds. Final libraries were purified by addition of 48 mL of AMPure XP beads and incubation for 5 min. Beads were washed twice with 70% ethanol, dried for 5 min, and eluted in 20 mL of water.