Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
ATAC-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
RS4:11 cells were electroporated alone (unedited, SPI1+/+) or with CAS9 and a guide RNA sequence (GGGGGUGGAAGUCCCAGUAA) targeting exon three of SPI1 (CRISPR Knockout Pool, Synthego). Editing efficiency approached 50%. Clonal populations of mono (SPI1+/-) and biallelically edited cells (SPI1-/-) were obtained via limiting dilution and clonal expansion. Edits were confirmed via Sanger sequencing and with PU.1 immunoblots. Cell aliquots of 100,000 were washed twice and then resuspended in cold lysis buffer before being spun down.  The supernatants were discarded, and then nuclei were resuspended in cold transposition reaction mix from the Illumina Tagment DNA Enzyme kit. The transposition reaction was incubated at 37°C for 45 min. Immediately following transposition, 10µl of 3M Sodium Acetate was added to the DNA which was then purified using a Qiagen MinElute Kit. Transposed DNA fragments were PCR amplified for 12 cycles using NEBNext High-Fidelity 2x PCR Master Mix(NEB) with Nextera primers (Illumina). The PCR reaction was cleaned up using AMPureXP beads (Agencourt)