GSM5830241: BulkATACseq_RANKL_rep_2; Mus musculus; ATAC-seq
GSM5830241
Cells were pelleted and resuspended in Recovery Cell Culture Freezing Medium (GIBCO) at -80°C to maintain native chromatin. At the start of the experiment, cells were thawed quickly at 37°C and washed twice with PBS. ATAC-seq libraries were prepared following an adapted protocol from Buenrostro et al., 2013 and Corces M.R., 2017. In brief, 50,000 cells per replicate were lysed in 50 µl of ice-cold ATAC Resuspension Buffer (RSB) containing 0.1% IGEPAL (Sigma), 0.1% Tween-20 (Sigma), and 0.01% Digitonin (Sigma) for 3 minutes on ice. Lysates were resuspended with 1 mL of cold ATAC-RSB containing 0.1% Tween-20 (Sigma) and nuclei were pelleted by centrifugation at 500 rcf for 10 minutes at 4°C in a fixed angle centrifuge. Nuclei were tagmented for 6 minutes at 37°C with in-house made Tn5 enzyme while shaking at 650 rpm. The tagmentation reaction was stopped with stop buffer (44 mM EDTA, 131 mM NaCl, 0.3% SDS, and 600 µg/ml proteinase K as final concentration). Genomic DNA was purified using 2x AMPure XP beads (Beckman Coulter) and subsequently amplified by PCR using KAPA HiFi Hotstart Ready Mix and Nextera Index Kit (Illumina) primers. Lastly, a reverse-phase 0.65x AMPure XP beads (Beckman Coulter) DNA purification step was done, followed by 1.5x AMPure XP beads (Beckman Coulter) purification. Library concentration was measured using the dsDNA Fluorescence Quantification Assays (DeNovix), and library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). DNA libraries were sequenced with an Illumina NextSeq 500, at a read length of 38 bp.