Illumina HiSeq 2500 sequencing; ATAC-Seq of Diversity Outbred mouse embryonic stem cells
ATAC-Seq of Diversity Outbred mouse embryonic stem cells
ESCs were derived from random blastocysts of Diversity Outbred (Jackson Laboratory #009376) mice using previously published protocols (Czechanski et al. 2014 Nature Protocols). As the ES cells expanded, they were transferred into 24 well plates, followed by 6 well plates, and finally 10cM dishes, all without feeder cells but in the same media. Cells were maintained in ES medium (ESM, 1i+LIF: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum, 100 U/mL Penicillin-Streptomycin, 2mM GlutaMAX, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 0.1mM 2-mercaptoethanol, approximately 2000U/ml LIF, and 3uM CHIR99021). For each mESC line, one 15cm dish of cells was grown to near confluence, washed 3x with PBS, and mechanically harvested to produce 80-150mg wet weight cell pellets (~50M cells). Cells from these pellets were cryopreserved. Cell lines were untreated. For ATAC-Seq we used the Fast-ATAC protocol (Corces et al., 2016 - https://www.ncbi.nlm.nih.gov/pubmed/27526324). Cryopreserved cells were thawed in a 37°C water bath, an aliquot of ~100,000 cells removed, then spun and washed with cold 50 µl PBS to remove DMSO. The cell pellet was resuspended in the 50 µl transposase reaction mix (25µl of 2X TD buffer, 2.5 µl of TDE1, 0.5 µl of 1% digitonin, and 22 µl of nuclease-free water). Transposition was carried out for 30 min at 37°C, and DNA purified using a Qiagen MinElute kit.  Libraries were amplified for a total of 9 cycles and purified using 1.7X AMPure beads. Nucleosome banding was visualized using the Agilent Tapestation.