title: GSM4998038: control_149; Homo sapiens; RIP-Seq
library_construction_protocol: Cells from three 10-cm plates were harvested, placed on ice, and crosslinked with 1% FA (Sigma) followed by crosslinking with 0.2 mg/ml AMT (Sigma; A4330) by irradiation with 350 nm UV light (Vilber Lourmat ECX.F20.L; 7 mW/cm2) for 45 min. After isolation of nuclei, 500 µl of MNase buffer (0.32 M sucrose, 50 mM Tris-Cl pH 7.5, 4 mM MgCl2, 1 mM CaCl2, and fresh 0.1 mM PMSF) was added. Chromatin and RNA were digested using 150 U of MNase (Worthington) for 15 min at 37 °C with shaking at 400 RPM on a thermomixer. The enzyme was inactivated by adding 0.8 mM EDTA. The samples were centrifuged at 12,000 g for 5 min, and the nuclei were sonicated using a Bioruptor at 40% amplitude with intervals of 2.2-sec pulses with 9.9-sec pauses for 12 min. This yielded DNA and RNA fragments of 100-500 bp in size. Antibodies used for IP were anti-pol II p-Ser2 (Abcam; ab5095), anti-pol II p-Ser5 (Abcam; ab5408), anti-U2 snRNP A (B-3) X (Santa Cruz; sc-393804X), and anti-HA (Abcam; ab9110). The samples were reverse crosslinked on ice by irradiation with 254 nm UV light for 10 min. RNA was extracted using Trizol reagent. (Sigma). Deep sequencing libraries were prepared using v2-pico (Takara Bio) library preparation kits. Sequencing of 50-bp single-end reads was performed using an Illumina HiSeq 2000.