GSM5115049: Stiff_3d_4; Sus scrofa; ATAC-seq
The omni ATAC libraries were prepared following the original omni-ATAC protocol (Corces 2017) with the following modifications. Cells were grown on hydrogels with each replicate having been grown and processed at different dates. Approximately 50,000 cells per well were used for each transposition reaction, with each reaction taking place while the cells were still attached onto their hydrogel substrate. A 300 μL total volume per 12-well plate well reaction was prepared as follows: 150 μL Tagment DNA Buffer (Illumina Ref 15027866), 7.5 μL Tagment DNA Enzyme 1 (Illumina Ref 15027865), 3 μL digitonin (Promega Ref G944A, diluted 1:1 with water), 30 μL Tween-20, 10.5 μL water, 89 μL PBS, 10 μL S2 Drosophila melanogaster nuclei; incubating for 50 minutes at 37°C. The Drosophila spike-in was added for the first replicate for a total of 500 nuclei (1% of total nuclei), and for the second replicate pair it was added for a total of 2,500 nuclei (5% of total nuclei). The pre-amplified transposed fragments were extracted from the hydrogel-attached cells with a phenol/chloroform/isoamyl alcohol (25:24:1) DNA precipitation. The post-amplified ATAC-libraries were cleaned-up with DNA Clean and Concentrator-5 Kit (Zymo Research Ref D4014). The qPCR amplification was done using NEBNext Ultra II Q5 Master Mix (NEB Ref M0544S), SYBR Gold (Life Tech Ref S11494), and Nextera DNA CD Indices (Illumina Ref 20015882). The libraries were size-selected to remove DNA fragments greater than 1000 bp with a Sage Science BluePippin. The ATAC-seq libraries were quantified with Qubit HS DNA assay and their fragment size-distributions determined with Agilent HS D5000 ScreenTape. Libraries were pooled and sequenced as paired-end 2x37 reads with a NextSeq sequencer at the University of Colorado Boulder BioFrontiers Next Generation Sequencing Core.