GSM5133016: polii_1h_no_A.rep1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed using anti-RNA Polymerase II antibody (Cell Signaling, 14958) as described previously 1. Briefly, following sorting, cells were re-suspended in 1ml pre-cooled Cell Lysis Buffer supplemented with protease inhibitors (PI) and PMSF for 20min on ice. Nuclei were pelleted and re-suspended in 1ml Nuclear Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS, fresh supplemented with PI and PMSF) for 10min on ice. 0.6ml IP dilution buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS, fresh supplemented with PI and PMSF) was added followed by sonication (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF) for 45min. Samples were pelleted at 15000rpm for 10min at 4℃ to remove cell debris. Supernatant was supplemented with 3.4ml IP dilution buffer fresh supplemented with PI and PMSF, 50g isotope-matched IgG and 50l protein A/G agarose beads (A:G = 1:1, ThermoFisher 15918014 and ThermoFisher 15920010) and rotated at 4℃ for 8h to preclear the chromatin. 200μl, chromatin was set aside as input chromatin. Precleared chromatin was then incubated with 35μl A/G agarose beads (A:G = 1:1) pre-bound with anti-RNA PolII antibody (5μg/IP) at 4℃ for overnight. Beads were washed once with IP wash buffer I (20mM Tris pH 8, 2mM EDTA, 50mMNaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20mMTris pH 8, 2mM EDTA, 500mMNaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer II (10mMTris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris pH 8, 1mM EDTA pH 8). All washing steps were performed on ice. Beads were moved to RT and eluted in 200μl fresh made Elution Buffer (100mM NaHCO3, 1%SDS). 12μl of 5M NaCl, 2μl RNaseA (10mg/ml) was added to IP and input samples and incubated at 65℃ for 2h, followed by addition of 3μl protease K (20mg/ml) and incubated at 65℃ overnight to reverse crosslinking. Finally, IP and input samples were supplemented with 10ul of 3M sodium acetate (pH 5.2), and DNA was purified with QIAquick PCR purification kit (QIAGEN 28106). ChIP-seq libraries were constructed using the Illumina's TruSeq ChIP sample preparation kit (Illumina, catalog no. IP-202-1012). Libraries were size selected using the SPRIselect beads (Beckman Coulter, catalog no. B23318) before PCR amplification. Libraries were then quantified through real-time PCR with the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Finally, libraries were pooled and sequenced on a Illumina NextSeq 500 platform using Illumina sequencing reagents.