title: GSM5233361: CIDOP_Input_rep2; Mus musculus; OTHER
library_construction_protocol: CIDOP chromatin capture reaction was performed as described in Mausser et al 2017, Epigenetics & Chromtain with a few adaptations. For MNAse digestion, 64U of MNAse from NEB per µg of DNA was used with 1mM CaCl2. MNAse reaction was placed directly at 37°C and incubated for 5min to obtain 10% of dinucleosomes and 90% of mononucleosomes. MNase reaction was stopped by adding 2mM EDTA + 30mM NaCl then samples were centrifuged at 13000g for 10min at 4°C. For CIDOP reaction, 60µg pre-clear mononucleosome-DNA was used with 0,5µM HiMID recombinant peptide in 500µL DP Buffer (16.7mM Tris-HCl pH=8, 167mM NaCl, 1.1% TritonX-100, 1.2mM EDTA). CIDOP retaction was incubated O/N at 4°C on a rotating wheel. Next day, 40µL Glutathione Sepharose 4B beads was added per IP then incubate at 4°C for 2h on a rotating wheel. IP reactions were washed as described in Mausser's protocole. For elution step, 100µL Elution Buffer and 3µL NEB Proteinase K were added and incubated at RT for 45min on a rotation wheel then 55°C for 1h. DNA was purified as described in Chromatin IP DNA purification Kit from Active Motif. For CIDOP-seq, CIDOP-DNA was eluted in 36µL with pre-warmed Elution Buffer then quantified with Qubit dsDNA HS Assay Kit. 1µL was also used to run Agilent High Sensitivity DNA electrophoresis to check DNA fragment size with majority of fragment at 150 nucleotides. 10ng of CIDOP-DNA was used for library preparation. 10ng CIDOP-DNA were used for library preparation. CIDOP-seq library preparation was performed as described in TruSeq ChIP Sample Preparation kit from Illumina with few adaptations. The Enrich DNA Fragment step is proceeded before Purify Ligation Products step. AMPure XP beads were air-dry 7min. 1µL library was diluted 1:10 in H20 to quantified library concentration by Qubit DNA HS assay quantification. Agilent High Sensitivity DNA Electrophoresis was run to check DNA fragment size. Before sequencing, ChIP-seq and CIDOP-seq libraries were quantified by qPCR. Libraries were sequenced 50bp or 100bp single-reads with 50 million reads of depth sequencing on NovaSeq 6000 Illumina machine at the MGX platforms.