GSM5605768: ATAC-seq_MDBK UT rep2; Bos taurus; ATAC-seq
30 minutes before harvesting, cells were treated with 200 U/mL DNase I (Worthington Biochemical #LS002006) for 30 minutes. ATAC-seq libraries were prepared according to Corces et al. 2017 (PMID: 28846090) using 50,000 viable (>90%) MDBK cells with modifications to the preamplification and qPCR steps. Purified post-transposition samples were initially amplified by combining 20 uL transposed sample, 2.5 uL 25 uM i5 primer, 2.5 uL 25 uM i7 primer, and 25 uL 2X NEBNext Ultra II Q5 Master Mix (NEB #M0554S). PCR cycling conditions: 30 s at 98°C, 5x(10 s at 98°C, 30 s at 63°C, 45 s at 65°C). qPCR was performed as follows: 5 uL of preamplified sample was combined with 0.5 uL 25 uM i5 primer, 0.5 uL 25 uM i7 primer, 3.75 uL nuclease-free water (Invitrogen #10977023), 0.25 uL 25X SYBR Gold (Invitrogen #S11494), and 5 uL 2x NEBNext Ultra II Q5 Master Mix. qPCR cycling conditions: 30 s at 98°C, 20x(10 s at 98°C, 30 s at 63°C, 45 s at 65°C). Preamplified samples were amplified for another 7 cycles. Final libraries were cleaned using a 0.5X/1.3X double-sided bead cleanup using KAPA Pure Beads (Roche #07983280001) and quantified using Qubit dsDNA High Sensitivity (Life Technologies #Q32851) and TapeStation 4200 HSD5000 (Agilent #50675588. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 (University of Colorado Genomics Core) as 150bp paired-end reads.