GSM6703858: ATAC-seq, Human LCL, DMSO, 1 hour, rep 1; Homo sapiens; ATAC-seq
The ATAC-seq libraries were made following the [Corces2017] (https://doi.org/10.1038/nmeth.4396) protocol. Briefly, after the 1 hour treatments, the 100,000 cells were transferred from their 12-well plate wells to 1.8 mL eppendorf tubes, and centrifuged at 500 x g for 7 minutes at 4°C. The supernatant was carefully removed and replaced with 50 μL of ice-cold lysis buffer (water with 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, 0.1% Tween-20, 0.01% Digitonin), the cells resuspended 4 times pipetting up and down, and incubated on ice for 5 minutes. Then, added 1 mL of wash buffer (water with 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) and the tubes inverted 4 times to mix. The tubes were centrifuged at 500 x g for 10 minutes at 4°C and the supernatant was carefully removed without disturbing the small cell pellet. The pellets were then carefully resuspended by pipetting 6 times with 50 μL of the transposition mix (25 μL Tagment DNA Buffer Illumina Ref. 15027866, 2.5 μL Tagment DNA Enzyme 1 Illumina Ref. 15027865, 0.5 μL Digitonin diluted 1:1 with water, 0.5 μL 10% Tween-20, 5 μL water, 16.5 μL PBS), and were incubated for 30 minutes in a heat block at 37°C, flicking the tube often. Afterwards, the samples were cleaned using the DNA Clean and Concentrator-5 Kit (Zymo Research Ref. D4014) following the manufacturer's instructions, and eluted in 21 μL elution buffer. Then, a PCR pre-amplification was done using NEBNext Ultra II Q5 Master Mix (NEB Ref. M0544S) using 5 cycles. Then, a qPCR was done using NEBNext Ultra II Q5 Master Mix, SYBR Gold (Life Tech Ref. S11494), and 5 μL of the pre-amplified sample, and the results used to determine the additional number of extra PCR cycles using Nextera DNA CD Indices (Illumina Ref. 20015882), which was just 1 additional cycle. The post-amplified ATAC-libraries were cleaned-up with the DNA Clean and Concentrator-5 Kit (Zymo Research Ref. D4014). The libraries were size-selected to remove DNA fragments greater than 1000 bp with a Sage Science BluePippin. The ATAC-seq libraries were quantified with Qubit HS DNA assay and their fragment size-distributions determined with Agilent HS D5000 ScreenTape. All samples were processed in parallel. After the samples were pooled and size-selected, they were cleaned-up using AMPure XP beads (Beckman Coulter Ref. A63881) at 1.5x volume and eluted into 20 μL of EB buffer (Qiagen Ref. 19086).