Rabbit anti-RNA Pol II Phospho-Ser2, Abcam, Cat# 5095
Sequenced DNA Library
Tissue was crosslinked 1% formaldehyde. The reaction was stopped with 0.125M glycine. After one wash with PBS, samples were lysed twice with L1 buffer (50mM Hepes pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 0.25% Triton X-100, 0.5% NP40, 10% Glycerol, protease inhibitors). The samples were incubated in L2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl), then lysed in L3 buffer (1M Tris-HCl pH8.0, 5M NaCl, 0.5 M EDTA, 0.5 M EGTA, 10% Na-Deoxycholate, 20% N-lauroylsarcosine), and sonicated with a Bioruptor (Diagenode). Sonicated chromatin was pre-cleared by incubating with Protein A Dynabeads for 2 hours. Pre-cleared chromatin was added to Dynabeads conjugated to specific antibodies and incubating overnight at 4°C, and 1 to 2.5% of chromatin were kept as input material. Antibodies used: DNMT3A (Santa Cruz 20703), H3.1/H3.2 (EMD Millipore ABE154), H3.3 (EMD Millipore 09-838), H2A.Z (EMD Millipore 07-594), H3K4me3 (EMD Millipore 07-473), H3K9me2 (Abcam ab1220), H3K9me3 (Abcam ab8898), H3K27me2 (Cell Signaling 9728), H3K27me3 (EMD Millipore 07-449), H3K36me3 (Abcam ab9050), H3 (Abcam ab1791), MECP2 (Chen et al., 2003), H3K27ac (Abcam ab4729), RNA Pol II (Abcam 817) and RNA Pol II Ser2P (Abcam 5095). Beads were washed twice with Low Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 150mM NaCl, 2mM EDTA), High Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 500mM NaCl, 2mM EDTA) and LiCl buffer (250mM LiCl, 1% NP40, 1mM EDTA, 10mM TrisHCl pH8.0, 1% Sodium Deoxycholate) at 4°C, and washed once with TE buffer at room temperature. Chromatin was eluted off the beads with TE+1%SDS at 65°C, and de-crosslinked by incubating overnight at 65°C. Samples were treated with RNaseA, and then with Proteinase K for 2 hours at 55°C. DNA was extracted by phenol-chloroform extraction and purified with a Minelute PCR cleanup column (Qiagen). Libraries were constructed by using Ovation Ultralow Library Systems (Nugen) following manufacturer instructions.