title: GSM8684886: HSV-1 RNaseH_DRIP-seq IP Rep1; Human alphaherpesvirus 1; OTHER
library_name: GSM8684886
library_construction_protocol: HSV-1 infected Jurkat cells were crosslinked with 1% paraformaldehyde (UP) for 10 minutes, followed by quenching with 2.5 M glycine (pH 6, final concentration 500 mM) for 5 minutes at room temperature. Lysis was performed using the lysis buffer from the NucleoSpin Tissue kit (Macherey-Nagel) at 65°C for 7 hours as per the kit protocol. Total nucleic acids were isolated using the NucleoSpin Tissue Kit and eluted in 100 µl of elution buffer (5 mM Tris-HCl, pH 8.5). The purified nucleic acid preparation was fragmented by sonication in 300 μl of Tris-HCl (pH 8.5, with 300 mM NaCl) for 2 cycles of 5 minutes (30 seconds ON, 30 seconds OFF, LOW setting, Bioruptor), yielding an average DNA fragment size of approximately 500 bp. Fragment analysis was performed using 1% agarose gel electrophoresis, with additional sonication applied if necessary. The sonicated DNA was purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) and eluted in 100 μl of elution buffer (5 mM Tris-HCl, pH 8.5). Twelve micrograms of DNA was diluted to a total volume of 100 μl with 5 mM Tris-HCl (pH 8.5), and 2% of the sample was retained as input DNA. Half of the sample was treated with 8 μl of RNase H (5000 U/ml; NEB) in a total volume of 80 μl at 37°C overnight. Dynabeads Protein A magnetic beads (ThermoFisher Scientific) were pre-blocked with PBS/5 mM EDTA containing 0.5% BSA. For antibody immobilization, 50 µl of pre-blocked Dynabeads Protein A was incubated with 10 μg of S9.6 antibody in IP buffer (50 mM Hepes/KOH, pH 7.5; 0.14 M NaCl; 5 mM EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2O) at 4°C for 4 hours with rotation. Six micrograms of genomic DNA was added to the antibody-bead mixture and gently rotated at 4°C overnight. The beads were then recovered and washed successively with 1 ml of the following buffers at 4°C, twice for each step: Lysis Buffer 1 (low salt): 50 mM Hepes/KOH (pH 7.5), 0.14 M NaCl, 5 mM EDTA (pH 8), 1% Triton X-100, 0.1% Na-Deoxycholate. Lysis Buffer 2 (high salt): 50 mM Hepes/KOH (pH 7.5), 0.5 M NaCl, 5 mM EDTA (pH 8), 1% Triton X-100, 0.1% Na-Deoxycholate. Wash Buffer: 10 mM Tris-HCl (pH 8), 0.25 M LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, 1 mM EDTA (pH 8). TE Buffer: 10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8). Elution was performed in 100 μl of elution buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA; 1% SDS) for 15 minutes at 65°C. Following purification using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), nucleic acids were eluted in 55 µl of elution buffer (5 mM Tris-HCl, pH 8.5). Illumina TruSeq ChIP Sample Preparation protocol were used to DRIP-seq. Purified ligation products were then amplified by PCR. Amplified libraries were sequenced (at the EMBL Genomics Core Facility, Heidelberg) using an Illumina HiSeq 2500 instrument in the single-end sequencing mode.