title: GSM8457886: NIH3T3_chrRNA-seq_FCS_15min_rep3; Mus musculus; RNA-Seq
library_construction_protocol: 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, 35 million 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. million 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. Chromatin RNA fraction was prepared from ∼20 million Raji cells. Cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl, pH 7.5, 10 mM NaCl, 0.5% NP40, and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2, and 10% Sucrose) was under-laid and then the nuclei were collected under 1,400 rcf centrifuge at 4°C for 5 min. Isolated nuclei were resuspended in 125 μl of NUN1 solution (20 mM Tri-HCl, pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol) followed by 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40). Fifteen minutes incubation was carried out on ice with mixing by max speed vortex for 5 s every ∼4 min and then chromatin pellets were precipitatd under 15,000 rcf centrifuge at 4°C for 10 min. Chromatin pellet was resuspended in 500 μl NUN2 buffer. Chromatin were precipitated under 15,000 rcf centrifuge at 4°C for 10 min, then Chromatin RNAs were extracted by 500 μl of Trizol reagent (Life technologies). The RNA extraction steps were repeated three times with rigorous treatment with TurboDNase in beetween. For DNase I sensitivity mapping, nuclei were prepared from 5 millions Raji cells per sample and incubated for 10 min at 37 °C with limiting concentrations of the DNA endonuclease DNase I (Sigma) supplemented with Ca2+ and Mg2+. A dose response experiment of 16 samples was performed with 2X dilution of the DNaseI between samples in cascade. Digestion was stopped by the addition of EDTA, and the samples were treated with proteinase K. The small fragments (<500 bp in length) were recovered on agrose gel from the sample in which a smear just started to appear using Quiagen gel purification kit and librairies were prepared using illumina Truseq kit. G4access was performed in K562 and NIH3T3 cells. Cells were pelleted and rinsed twice in phosphate-buffered saline buffer (PBS). For each experiment, 5 millions cells per titration point were re-suspended in 50 μL of prewarmed permeabilization buffer (150 mM of sucrose, 80 mM KCl, 5 mM KH2PO4, 5 mM MgCl2, 0.5 mM CaCl2 and 35 mM HEPES pH 7.4) supplemented with 0.2% (v/v) NP40 and incubated for 5 minutes at 37ºC prior digestion. MNase digestions, were then performed by adding a volume of 500 μL of prewarmed MNase reaction buffer (150 mM sucrose, 50 mM Tris-HCl pH 8, 50 mM NaCl and 2 mM CaCl2) supplemented with either 3, 6, 12, 25 or 50U of MNase (Merck, 10107921001). Digestions were incubated at 37ºC for 10 min and stopped on ice and by adding 11 μL of 500 mM EDTA to each reaction. Samples were then incubated 10 minutes on ice with 550 μL of SDS lysis buffer (1% (v/v) SDS, 10 mM EDTA and 50 mM Tris.HCl pH 8). Before DNA purification, 1 mL of water was added to dilute the SDS and the samples were incubated with 5 µL of RNAse A (ThermoFisher, EN0531) at 37 ºC for 2 hours and with 8 µL of proteinase K (Euromedex, 09-0911) at 56 ºC for 2 hours to complete the lysis. To then quality control the MNase digestions: 125 μL of each sample were cleaned-up using QIAquick PCR Purification Kit (QIAGEN, 28106) and assessed by agarose gel and Bioanalyzer. At this step, for efficient G4access, samples should present ~30% (+/-5%) of mono-nucleosomes. Importantly, this assessment should be performed on purified DNA that does not contain the subnucleosomal fraction, using a bioanalyzer equipment. The remaining of the samples was then purified by phenol-chloroform and ethanol precipitation for subsequent steps. We recommend that, when implementing this method, a wide range of MNase concentrations shall be tested in a first round of preparative experiments to narrow down the condition in which the critical fraction of 30% of mononucleosome shall be obtained. Our experiences showed this fraction is on average optimal for best G4 sequence recovery. The 0-100 bp size-selected fragments from MNase digestions that have ~30% of mono-nucleosomes were subjected to DNA library preparation. In parallel, genomic DNA libraries were sonicated by Bioruptor® Pico sonicator (Diagenode) to obtain DNA fragments of ~150 bp to be used later as reference data sets for bioinformatic analyses. Paired-end libraries were constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, E7645S) using a starting material of 50 ng. DNA fragments were treated with end-repair, A-tailing and ligation of Illumina-compatible adapters. Clean-up of adaptor-ligated DNA was performed by using CleanNGS beads (CNGS-0050) with a bead:DNA ratio of 2:1. The purified products were amplified with 8 cycles of PCR. Finally, samples were cleaned up with a bead:DNA ratio of 0.8:1 to remove the free sequencing adapters.  TT-seq experiments were performed in biological duplicates using 47 millions Raji cells treated with 10 μM pyridostatin (PDS; Sigma SML0678) or H2O (Control) for 10 minutes. Newly synthetised RNAs were labelled with 500µM 4-Thiouridine (4SU; Sigma T4509) for 10min at 37°C at the time of the treatment. In parallel, 60 x106 drosophila S2R+ cells were labelled for 10min with 500µM 4SU. Cells were then harvested by centrifugation and Total RNA was isolated with QIAzol reagent (QIAGEN) according to the manufacturer's instructions. Drosophila RNA was used as spike-in and added to Raji TotRNAs (1:40 ratio). TotRNAs were fragmented (two-times 105µg in 120µL) using a Covaris sonicator M220 (7°C, Power 75W, Duty factor 1%, burst 200) to generate RNA fragments of 200bp to 10kb. 4SU-labeled RNA from 210µg of Fragmented RNA was biotinylated, pulled-down using streptavidin beads and eluted with freshly prepared 100mM DTT. 4sU-labeled RNA (TT-seq) were treated with RNase-free DNase I, purified with miRNeasy MinElute spin column and eluted in 15µL Nuclease-free water. Prior to library preparation, samples were quantified with Nanodrop and quality was assessed by Bioanalyzer Nano RNA ChIP. Ribosomal RNA were depleted from samples using NEBNext rRNA Depletion Kit V2 (NEB ; E7400) and sequencing libraries were prepared with NEBNext Ultra II Directional Kit (NEB, E7760) according to manufacturer's instructions. ChIP-seq, RNA-seq Mnase-seq, DNase-seq and ChIP-seq librairies were prepared using Illumina's ChIP Sample Prep Kit according to standard protocol. ChrRNA-seq librairies were prepared using Illuminas's "TruSeq® Stranded Total RNA Library Prep Gold (ref. 20020599)" kit according to standard protocol. TT-seq librairies were prepared using NEBnext Utra II Directional Kit.