GSM6448496: HL-60 DMSO-Treated acH2A.Z ChIP-Seq; Homo sapiens; ChIP-Seq
Nascent RNA/DNA capture was performed using Click-iT® Nascent RNA Capture Kit (ThermoFisher) according to the manufacturer's instructions with minor modifications. Briefly, 1. Labeling the cells with EU/EdU. 200 mM EU or 30 mM EdU stock solutions were added to the cells, to a final concentration 0.5 mM or 30 uM, respectively. 2. Incubation. The cells were incubated for 1 or 2 hours. 3. RNA/DNA isolation. The cells were harvested and the RNA/DNA were isolated and dissolved in 14 uL of H2O. 4. Biotinylation of RNA/DNA by Click reaction. Click-iT reaction cocktail (50 uL per reaction) was prepared accordingly to manufacturer's instructions: a mixture containing 1x Click-iT EU buffer; 2 mM CuSO4; 1 mM Biotin azide; 13.25 uL of the isolated RNA; 10 mM Click-iT® reaction buffer additive 1; 12 mM Click-iT reaction buffer additive 2 was prepared. After adding each component, the reaction cocktail was gently mixed by vortexing. The addition of the Click-iT reaction buffer additive 1 stock initiates the click reaction between the EU-RNA/EdU-DNA and biotin azide. Afterwards the Click-iT reaction buffer additive 2 is added and incubated for 30 minutes with gentle vortexing. 5. RNA/DNA precipitation. 1 uL of UltraPure Glycogen, 55 uL of 7 M ammonium acetate, and 750 uL of chilled 100% ethanol were added to the click reaction, incubated at –70°C for at least 30 minutes and after centrifugation the pellet was dissolved in 125 uL of H2O. 6. Binding biotinylated RNA/DNA to Dynabeads MyOne Streptavidin T1 magnetic beads. The RNA/DNA binding reaction mixture included: 125 uL 2xClick-iT RNA binding buffer; 2 uL Ribonuclease Inhibitor or 2 uL of water for DNA; 125 uL of the isolated biotinylated RNA/DNA. The RNA binding reaction mixture was heated at 68–70°C for 5 minutes and 50 uL of bead suspension added into the heated RNA binding reaction mixture. The tube containing the RNA/DNA binding reaction was incubated at r.t. for 30 min while gently vortexing to prevent the beads from settling. The beads were immobilized using the magnet and washed 5 times with 500 uL of Click-iT reaction wash buffer 1 and 5 times with 500 uL of Click-iT reaction wash buffer 2. Finally, the beads were resuspended in 50 uL of Click-iT reaction wash buffer 2 and the captured RNA immediately processed to cDNA synthesis. The captured DNA was released into 50 uL of boiling water and used in qPCR analyses. RIP-Seq - Immunoprecipitated RNA were processed for sequencing as described by Di Ruscio et al.(Di Ruscio et al., 2013) with some modifications. RNAs were depleted of ribosomal RNA with Ribo-ZeroTM Magnetic Gold Kit. Double stranded cDNA libraries were constructed using ScriptSeq v2 RNA-Seq Library Preparation Kit. The libraries were subjected to final size-selection in 3% agarose gels. 250-500 bp fragments were excised and recovered using the Qiaquick Gel Extraction Kit (Qiagen). RNA-Seq - RNA was extracted with TRI Reagent (MRC). RNA samples were treated with DNase I (10 U of DNase I (Roche) per 3 ug of total RNA; 37ºC for one hour; in the presence of RNase A inhibitor).   RIP-Seq - Immunoprecipitated RNA were processed for sequencing as described by Di Ruscio et al.(Di Ruscio et al., 2013) with some modifications. RNAs were depleted of ribosomal RNA with Ribo-ZeroTM Magnetic Gold Kit. Double stranded cDNA libraries were constructed using ScriptSeq v2 RNA-Seq Library Preparation Kit. The libraries were subjected to final size-selection in 3% agarose gels. 250-500 bp fragments were excised and recovered using the Qiaquick Gel Extraction Kit (Qiagen). RNA-Seq - RNA was extracted with TRI Reagent (MRC). RNA samples were treated with DNase I (10 U of DNase I (Roche) per 3 ug of total RNA; 37ºC for one hour; in the presence of RNase A inhibitor). RNAs were depleted of ribosomal RNA with Ribo-Zero Magnetic Gold Kit. Double stranded cDNA libraries were constructed using ScriptSeq v2 RNA-Seq Library Preparation Kit. The libraries were subjected to final size-selection in 3% agarose gel. 250-500 bp fragments were excised and recovered using the Qiaquick Gel Extraction Kit (Qiagen). Libraries were sequenced on a Hi-Seq-2000 Illumina. ChIP-Seq – ChIP libraries' construction was performed as described (Mikkelsen et al., 2008) and paired-end sequenced on NextSeq500 platform, at a reading length of 36 nucleotides.