title: GSM7669299: YCCEL1 DMSO2; Human gammaherpesvirus 4; ATAC-seq
library_name: GSM7669299
library_construction_protocol: 1 × 105 cells (>95% viability) were washed in 50 ml cold PBS, spun down at 500 x g and 4°C for 5min Cells were resuspended in 50 ml cold ATAC-Resuspension Buffer (RSB) (10 mM Tris-HCl, pH7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% IGEPAL CA-630, 0.1% Tween-20 and 0.01% Digitonin. Resuspended cells were kept on ice for 3 min, then washed with 1 ml cold ATAC-RSB containing 0.1% Tween-20 but no IGEPAL CA-630 or Digitonin.  Pellet nuclei at 500 x g and 4°C for 10 min, and the supernatant was removed. The pellet was then resuspended in a 50 ml Tn5 transposase reaction mixture following the manufacturer's protocol (Illumina Tagment DNA Enzyme and Buffer, Illumina) and incubated at 37°C for 30 min in a thermomixer with 300 rpm mixing. DNA was purified using a MinElute PCR purification kit (Qiagen) and eluted in 10 ml Elution Buffer for library amplification.  PCR amplification of fragmented DNA was done using the NEBNext HiFi PCR mastermix (New England Bioloabs) with a universal forward and sample-specific reverse oligo for sample barcoding using the following PCR conditions: initial incubations of 72°C for 5 min and 98°C for 30 s, followed by 5 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min.  Additional number of cycles was determined for each sample through a “side” qPCR reaction using an aliquot of the PCR as template to determine the number of cycles needed to reach 1/3 of the max fluorescence.  PCR products were run on a 1% agarose gel, regions from ~50 bp to ~1 kb were excised, and DNA was extracted using a gel extraction kit (Qiagen).