GSM9865096: BJ fibroblast, Targeted ATAC-see, LaminB1, high-salt (300mM NaCl); Homo sapiens; ATAC-seq
Following imaging on a Nikon Eclipse Ti2 inverted microscope as described above, reverse cross-linking was performed as in and performed as in (Kirkland et al., In press). Briefly, HBSS was aspirated and replaced with a reverse-crosslinking solution adapted from (Chen et al., 2016a) consisting of 50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 0.2 M NaCl and 250 ug/ml proteinase K. Cells were physically detached using a cell scraper, transferred to microcentrifuge tubes, and incubated overnight at 65°C with agitation at 1,000 rpm in a thermomixer. DNA was subsequently purified using the MinElute Reaction Cleanup Kit (Qiagen, Cat#: 28204) according to the manufacturer's instructions. Eluted DNA was amplified using 11, 14, and 17 PCR cycles with NEBNext® HiFi 2× PCR Master Mix (NEB, M0541S) and pre-mixed Illumina DNA/RNA UD Indexes (Set A; cat. no. 20091646), following the PCR protocol described in (Buenrostro et al., 2013). Library quality was assessed by Agilent TapeStation, and products from all three cycle numbers were pooled and purified using 1.8X SPRI beads (Beckman Coulter, cat. no. B23318). Sequencing was performed on an Illumina NextSeq 2000 (P3 flow cell) with paired-end 50 + 50 bp reads and 10 bp dual-indexing (50/10/10/50 cycle configuration).