GSM8464728: HEK293T_DTAG_rep13; Homo sapiens; ATAC-seq
At the time of harvest, cells were moved to ice and scraped in 1mL ice cold PBS, and 100μL (∼5 x 10^4 cells) were transferred to 1.5 mL tubes. Cells were centrifuged at 500 x g for 5 minutes at 4°C, and the pellets were resuspended in 50 μL cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin, adjusted to pH 7.4) and incubated on ice for 3 minutes. Samples were washed with 1 mL cold wash buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20). Cells were centrifuged at 500 x g for 10 minutes at 4°C, and pellets were resuspended in the transposition reaction mix (25 μL 2X TD buffer (Illumina), 2.5 μL TDE1 Tn5 transposase (Illumina), 16.5 μL PBS, 0.5 μL 1% Digitonin, 0.5 μL 10% Tween-20, 5 μL nuclease-free water) and incubated in a thermomixer at 37°C and 100rpm for 30 minutes. DNA was extracted with the DNA Clean and Concentrator-5 Kit (Zymo Research). Sequencing adapters were attached to the transposed DNA fragments using NEBNext Ultra II Q5 PCR mix (New England Biolabs), and libraries were amplified with 8 cycles of PCR. PEG-mediated size fractionation (Lis 1980) was performed on the libraries by mixing SPRIs-elect beads (Beckman) with each sample at a 0.5:1 ratio, then placing the reaction vessels on a magnetic stand. The right side selected sample was transferred to a new reaction vessel, and more beads were added for a final ratio of 1.8:1. The final size-selected sample was eluted into nuclease-free water. This size selection protocol was repeated to further remove large fragments.