GSM4824647: Raji_Mnase-seq_T30_PDS1_1; Homo sapiens; MNase-Seq
Fifty million cells were used to perform each Pol II ChIP-seq experiments. Cells were crosslinked for 10 min at 20°C with the crosslinking solution (10 mM NaCl, 0.1 mM EDTA pH 8, 0.05 mM EGTA pH 8, 5 mM HEPES pH 7.8 and 1% formaldehyde). The reaction was stopped by adding glycine at a final concentration of 250 mM. After 5 min of formaldehyde quenching, cells were washed twice with cold PBS and resuspended in cold 2.5L LB1 (50 mM HEPES pH 7.5, 140 mM NaCl,1 mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100) at 4°C for 20 min on a rotating wheel. Nuclei were pelleted down by spinning at 1350 rcf in a refrigerated centrifuge and washed in 2.5L LB2 (200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 10 mM Tris pH 8) for 10 min at 4°C on a rotating wheel followed by centrifugation to collect nuclei. Nuclei were then resuspended in 1L LB3 (1 mM EDTA pH 8, 0.5 mM EGTA pH8, 10 mM Tris pH8, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using Bioruptor Pico (Diagenode) in 15L tubes for 20 cycles of 30 s ON and 30 s OFF pulses in 4°C bath. All buffers (LB1, LB2 and LB3) were complemented with EDTA free Protease inhibitor cocktail (Roche), 0.2 mM PMSF and 1 µg/L Pepstatin just before use. After sonication, Triton X-100 was added to a final concentration of 1% followed by centrifugation at 20000 rcf and 4°C for 10 min to remove particulate matter. For sequencing of nucleosomal DNA in Raji cells, 35x106 cells were resuspended in 350μl Solution I (150 mM sucrose, 80 mM KCl, 5 mM K2HPO4, 5 mM MgCl2, 0.5 mM CaCl2, 35 mM HEPES pH 7.4) and NP40 was added to a final concentration of 0.2%. Cell membranes were permeabilized for five min at 37°C. MNase was prepared at 50, 25, 12, 6 or 3 units in 0.5L of Solution II (150 mM sucrose, 50 mM Tris pH 8, 50 mM NaCl, 2 mM CaCl2) and incubated with 50 µL of cellular preparation, corresponding to 5.106 cells, for exactly 10 min at 37°C. The reactions were stopped by adding of EDTA to a final concentration of 10 mM. The cells were lysed using 1.45L of SDS Lysis Buffer (1% SDS, 10 mM EDTA pH 8, 50 mM Tris pH 8), with 10 min incubation at 4°C. 200μl aliquots were taken for purification and the remaining extracts were stored at -80°C. An equal volume of TE (200ul) was added to the aliquots, followed by subsequent 2 hour treatments with each 0.2µg/L final concentrations of RNase A and Proteinase K at 37°C and 55°C, respectively. DNA was extracted by two subsequent phenol:chloroform:isoamylalcohol (25:24:1) extractions, further purified using QIAquick PCR purifications colums (Qiagen, Germany). Nucleosomal digestion was verified by running 500ng of DNA on a 1.5% agarose gel as well as on DNA high-sensitivity 2100 Bioanalyzer chips (Agilent, USA). Digestions showing 75% of mononucleomes (running at 150bp) were selected for library preparations. ChIP-seq Mnase-seq and ChIP-seq librairies were prepared using Illumina's ChIP Sample Prep Kit according to standard protocol.