title: GSM6745539: H3K27ME3, SP140 ko, replicate 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Daudi and Ramos cells were harvested and washed twice with PBS. Then, cells were crosslinked by formaldehyde 1% for 10 min at room temperature under rotation. Crosslinking reaction was quenched by adding Glycine (0.125 M final) to the solution. Crosslinked cells were then washed twice with PBS and lysed with SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH8, 5 mM EDTA, 0.5% SDS). Cells were incubated on dry ice for 10 min and then pellet at 1200 rpm for 6 min. Cells were resuspended in ice cold IP Buffer (IP Buffer: 1 volume of SDS Buffer: 0.5 volume of Triton dilution Buffer (100 mM Tris HCl pH8.6, 100 mM NaCl, 5 mM EDTA, 5% Triton). Fixed cells were then sonicated to an average length of 200-500 bp by Branson Sonifier 250 (settings: output control 1, duty cycle 30) for 6 times on ice with 2-3 min rest in between. The quality of DNA sharing was assessed by loading samples on agarose gel (2%) and using Agilent Bioanalyzer instrument. Then, sonicated chromatin was centrifuged at 20000g for 30 min at 4oC. Sonicated chromatin was quantified by Bradford and used to immunoprecipitate Sp140, H3K27Me3 and MBD3 overnight at 4oC. IgG were used as negative control while input (1%, was saved as total control to normalize the signal). 40 μL of G-Sepharose beads were added and incubated for 4 hours. Then, beads were washed 3 times with washing buffer 1 (1% Triton, 0.1% SDS, 150 mM NaCl, 2 mM EDTA pH8, 20 mM Tris-HCl pH8) and once with washing buffer 2 (1% Triton, 0.1% SDS, 500 mM NaCl, 2 mM EDTA pH8, 20 mM Tris-HCl pH 8). Samples were decrosslinked by adding the decrosslinking solution (1% SDS, 0.1M NaHCO3) and incubated overnight at 65oC. Libraries were constructed using the ChIP TruSeq Chip Kit (Illumina) sequencing library preparation kit. Sequencing was performed on Illumina NextSeq 500, in the following mode: 75nt paired-end in order to obtain 30M clusters of fragments for Sp140 ChIP-Seq libraries and 60M clusters of fragments for H3K27me3 ChIP-Seq libraries. Each size selected library was diluted according to the final concentration of 11 pM and clonally amplified using the Ion Proton™ Hi‑Q™Template Kit (Thermo Fisher Scientific/Life Technologies) with IonOneTouch 2 instrument (Thermo Fisher Scientific/Life Technologies). After emulsion PCR, DNA positive ISPs were recovered and enriched according to standard protocols with the IonOneTouch ES Instrument (Thermo Fisher Scientific/Life Technologies). A sequencing primer was annealed to DNA positive ISPs and the sequencing polymerase bound, prior to loading of ISPs into Ion P1 sequencing chips. Sequencing of the samples was conducted according to the Ion Proton™ Hi-Q Sequencing Kit Protocol. One P1 sequencing chips with the 3 libraries were loaded and run on an Ion Proton sequencer to generate reads with an average length of 150-200.