GSM6448502: HL-60 RIP-Seq acH2A.Z; Homo sapiens; RIP-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. Nanopore RNA-Seq - Total nuclera RNA was  isolated after one hour from the release of the S-phase using TRI Reagent (MRC) according to manufacturer's istructions. RNA sample was  depleted of ribosomal RNA with Ribo-ZeroTM Magnetic Gold Kit. The Polyadenylated RNAs were enriched using oligod(T)25 magnetic beads (NEB S1419S). The remaining polyA minus RNAs wer polyadenylated using TdT (NEB M0315S). The resulting RNA from both fractions, polyA plus and minus were subjected to library preparation using the Direct RNA sequencing kit (SQK-RNA002, Oxford Nanopore Technologies, UK)  according to manufacturer's istructions. Libraries were sequenced on PromethIon device using R10.4 flow cells.