title: GSM3728279: siC-Pol2_S2P; Homo sapiens; ChIP-Seq
library_construction_protocol: KAPA Hyper Prep Kit (KK8502, KapaBiosystems) For chromatin PAR-CLIP, chromatin extraction was performed based on a published protocol (Brugiolo et. al., Nucleic Acids Research, 2017, 45(18):10452–65). The chromatin pellet from 3* 15 cm of HEK293T cells was solublized 900 µL chromatin extraction buffer (20 mM Tris-Cl [pH 7.5], 100 mM KCl, 2 mM MgCl2, 2.5 mM CaCl2, 0.3 M sucrose, and 0.1% v/v Triton X-100, 1% protease and phosphatase inhibitor) with 5 U/mL Micrococcal Nuclease (N3755, Sigma) by shaking at 4 ºC for 2 hours. The immunoprecipitation was performed with 5 ug of rabbit anti-hnRNPG antibody (Ab190352, abcam) overnight and 100 uL Protein A beads (10002D, Thermo) for another 4 hours at 4ºC. Co-immunoprecipitated RNA fragments were then washed, end-repaired on-beads, size-selected by SDS-PAGE, and released by Protease K digestion. RNA was purified by Acid-Phenol:Chloroform extraction and overnight ethanol precipitation. For ChIP-seq, we followed the protocol of ChIP-IT® Express kit (53008, Active motif) with Spike-in normalization strategy. The chromatin samples were sheared by sonication into 200-500 bp in size. For each immunoprecipitation reaction, 20 ug of the sample chromatin, 5 ug of the antibody, 10 ng of Spike-in chromatin (53083, Active motif), and 1 ug of Spike-in antibody (61686, Active Motif) were used. After washing, reverse crosslinking, and treatment with RNase A and Protease K, the DNA was purifed by with DNA Clean & Concentrator-5 (D4013, Zymo research).