GSM5315897: WT-EV-H3K36me2-rep2; Mus musculus; ChIP-Seq
ChIP-seq: DNA was purified with QIAquick PCR purification kit (Qiagen) according to manufacturer's instructions. DNA was purified with QIAquick PCR purification kit (Qiagen) according to manufacturer's instructions. DNA libraries were prepared with 2–10 ng of DNA using an in-house protocol (Blecher-Gonen et al., 2013) by the IEO genomic facility and sequenced on an Illumina HiSeq 2000. RNA-seq: RNA-seq was performed following SMART-seq2 protocol (Picelli et al., 2014)  with minor modifications. Briefly, poly-A containing mRNA molecules obtained from 1 μg of total RNA were copied into first-strand cDNA by reverse transcription and template-switching using oligo (dT) primers and an LNA-containing template-switching oligo (TSO). Resulting cDNA was pre-amplified with KAPA HotStart Taq enzyme (Kapa Biosystems) and then purified with Ampure beads (Agencourt AMPure XP- Beckman Coulter). One nanogram of pre-amplified cDNA was tagmented with in-house produced Tn5 transposase and further amplified with KAPA HotStart Taq enzyme. After purification with Ampure beads, the quality of the obtained library was assessed by Bioanalyzer (High Sensitivity DNA kit, Agilent Technologies), prior to sequencing. Each RNA-seq experiment was performed in triplicate. Hi-C: In situ Hi-C was performed as described (Rao et al., 2014) with slight alterations to the protocol. 5x10^6 ESCs were crosslinked for 10 minutes in 1% formaldehyde before quenching in 125mM Glycine for 5 minutes. Cell pellets were washed twice in PBS before contact generation. Pellet was resuspended in 500uL Hi-C lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% NP40 and protease inhibitors) and incubated with rotation for 30 minutes at 4°C. Cells were pelleted and washed in 500uL Hi-C lysis buffer. Pellet was resuspended in 100uL 0.5% SDS and incubated for 10 minutes at 62°C. 285uL of H20 and 50uL of 10% Triton X-100 was added to quench the SDS at 37°C for 15 minutes. 50uL of 10xNEB DpnII buffer and 750U of DpnII were added (NEB R0453M) then incubated at 37°C overnight with shaking. Digestion was heat inactivated the next day for 20 minutes at 62°C. Biotin fill in was performed by addition of 52uL fill-in mix (0.288mM dCTP/dTTP/dGTP, 50U DNA Polymerase I, Large Klenow fragment (NEB M0210) and 0.288mM biotin-dATP (Thermo 19524016)). Fill-in reaction was incubated at 37°C for 1 hour with shaking. Ligation was performed by adding 150uL 10x NEB T4 DNA ligase buffer, 125uL 10% Triton X-100, 3uL 50mg/mL BSA and 8000U of T4 DNA ligase and H20 up to 1.5mL. Ligation was performed overnight at 25°C with shaking. Nuclei were pelleted before resuspending in IP buffer (33 mM Tris-HCl pH 8, 100 mM NaCl, 5 mM EDTA, 0.2% NaN3, 0.33% SDS, 1.66% Triton X-100) and sonicating to fragment size between 200-1000bp. 15uL of the sample was de-crosslinked overnight at 65°C with 85ul of decrosslinking buffer (0.1M NaHCO3, 1% SDS) before clean up using QiaQuick PCR clean up kit from Qiagen. Library prep was performed as described (Mumbach et al., 2016). Streptavidin C-1 beads (Thermo 65001) were washed and resuspended in 2x biotin binding buffer (10mM Tris-HCl pH7.5, 1mM EDTA, 2M NaCl). 10ng of DNA was used for biotin capture, streptavidin C-1 beads were added and incubated for 15 minutes at room temperature with shaking. Beads were washed twice at 55°C for 2 minutes in Tween wash buffer (5mM Tris-HCl pH7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween). Beads were washed once in 1xTD buffer (20mM Tris-HCl pH7.5, 10mM MgCl2, 20% DMF) before being tagmented with in-house produced Tn5 transposase in TD buffer for 55°C for 10 minutes. TN5 was quenched in 50mM EDTA for 30 minutes at 55°C before washing twice in 50mM EDTA, twice in Tween wash buffer and once in 10mM Tris. PCR amplification was carried out on beads using KAPA HotStart Taq enzyme. After purification of PCR product with Ampure beads, the quality of the obtained library was assessed by Bioanalyzer (High Sensitivity DNA kit, Agilent Technologies), prior to sequencing.