GSM7844641: NOK siLEF1 FL+DN H327ac rep 2; Human gammaherpesvirus 4; OTHER
CUT&RUN experiments were performed using the CUT&RUN Assay kit (#86652) from Cell Signaling Technology (protocol no. 1884). Native chromatin-transcription factor footprint analysis was performed without prior crosslinking. Histone acetylation analysis of NOKs-Akata included formaldehyde crosslinking for 90 seconds with 0.1% formaldehyde followed by quenching with 135 mM Glycine for 7 minutes prior to Accutase treatment and lifting. 150,000 Akata BX1 or 250,000 EBV+NOKs per sample were washed twice in kit wash buffer. Cells were then captured with Concanavalin A magnetic beads (10 µL/sample) and re-suspended in the kit Antibody buffer at a total volume of 100 µL/sample. 500 ng of primary antibody was then added to the 100 μL sample and incubated for 12 hours at 4°C. Cell:Bead suspensions were then centrifuged at 200 x g for 1 min followed by magnetization and resuspended in 500 µL kit wash buffer for 2 X wash cycles. Following thorough washing, Cell:Beads were re-suspended in 100 µL wash buffer containing 0.2 mg of a donkey-anti rabbit secondary antibody (A16037, Thermo) and were incubated 1 hour at 4°C. Samples were then washed 2 X as described. Binding of pAG-micrococcal nuclease was performed following re-suspension of Cell:Beads in 50 µL wash buffer and supplemented with 1.5 µL pAG-MNase followed by incubation at 4°C for 1 hour. Cell:Beads were then thoroughly washed 2X times in wash buffer using centrifugation at 200 x g and magnetization. Cell:Beads were resuspended in 150 µL wash buffer and supplemented with 3 µL 100 mM CaCl2 at 4°C for 30min to promote cleavage of DNA.  Cleavage reactions were halted following addition of 150µL kit STOP Buffer containing S. cerevisiae Spike-In DNA (50 pg/sample) for sample normalization. Samples were incubated at 37°C for 20 min to release cleaved fragments, followed by centrifugation at 16,000 x g for 5 min enriched chromatin fractions were then collected following sample placement on magnet strip. 2 µL 10% SDS and 1.5 µL Proteinase K (20 mg/mL) was added to each sample and incubated for 1 hr at 50°C. CUT&RUN DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction. 300 µL PLC was added to enriched chromatin and vortexed followed by centrifugation at 16,000 x g for 5 min at room temperature. 300 µL chloroform was added to the centrifuged samples and inverted 10 X followed by centrifugation at 16,000 x g for 5 min at room temperature. The top liquid phase was then removed and transferred to a clean tube containing 2 µL 2 mg/mL Glycogen as carrier. 500 µL 100% ethanol was then added followed by vortexing and incubation at -80 °C for 1 hour. Samples were then centrifuged at 16,000 x g for 20 min at 4°C. Ethanol was removed and DNA pellets washed in additional 500 µL 100 % ethanol. Samples were re-centrifuged, ethanol was removed and pellets allowed to dry. CUT&RUN DNA pellets were dissolved in 50 μL 0.1× TE (1 mM Tris-HCl pH 8.0, 0.1mM EDTA) in preparation for down stream analysis. Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) with protocol modifications described in (Kong NR, Chai L, Tenen DG, Bassal MA. A modified CUT&RUN protocol and analysis pipeline to identify transcription factor binding sites in human cell lines. STAR Protoc. 2021;2(3):100750). Samples were indexed with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, E7600). 4-6 ng of CUT&RUN DNA was processed for each sample. DNA was end repaired and adaptors were ligated. To clean up the reaction, 1.75× volume of Agencourt AMPure XP beads was added to retain short ligation products. PCR amplification was performed for 15 cycles. The resulting libraries were purified with 1.2× volume of AMPure beads then analyzed and quantified by Tapestation (D1000 screen Tape). Libraries were normalized to 4 nM and pooled. The library pool was denatured and diluted to approximately 12 pM and spiked with 10% PhiX as an internal control. Illumina paired-end sequencing (80x80 cycles) was performed using either on the MiSeq with the MiSeq Reagent kit v3 (150 cycles) or on the Nextseq 500 platform with NextSeq 500/550 High Output Kit v2 (150 cycles).