title: GSM6456048: U2OS_ChIPseq_Dyskerin_flavopiridol_rep2; Homo sapiens; ChIP-Seq
library_name: GSM6456048
library_construction_protocol: 6 to 10 million cells were crosslinked for 5 minutes at room temperature with 16% methanol-free formaldehyde (Thermo Fisher Scientific) used at a final concentration of 1%. After quenching the reaction with 125 mM glycine final concentration, cells were washed twice in ice-cold PBS and collected by scraping. Pellets were resuspended in buffer A (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 substitute) and incubated on ice for 10 minutes. After centrifugation (5000 rpm for 5 minutes at 4°C), the supernatants containing the cytoplasm were removed and the nuclei-containing pellets were washed in buffer A without detergent. Pellets were then resuspended in shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and sonicated with a ME220 focused-ultrasonicator (Covaris). After clarification by centrifugation (13000 rpm for 10 minutes at 4°C), the supernatant was adjusted in composition to RIPA buffer and transferred to a new tube to be used for immunoprecipitation. Equal amounts of chromatin (as measured by DNA concentration) were incubated rotating overnight at 4°C with pre-conjugated beads (20 μl of Dynabeads and 3 μg antibody, dyskerin (Abnova, H00001736-m04) or RNAPII (MBL, MAB0601)) in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Immunoprecipitates were washed once with each of the following: RIPA buffer, RIPA buffer high salt (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LCiCl, 0.5% NP-40 substitute, 0.5% sodium deoxycholate), and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The beads were then resuspended in TE buffer with 0.5% SDS supplemented with proteinase K (New England Biolabs) and RNase A (Thermo Fisher Scientific) and incubated with shaking at 65°C for 4 hours. The DNA was recovered by phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation using standard procedures, and resuspended in TE buffer.  Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions.