GSM7090924: ATAC_HNF1B_CRISPRi_enh2; Homo sapiens; ATAC-seq
Adherent cells were scraped from culture dishes and centrifuged at 500 × g for 5 min. 10,000 cells were suspended in 50 μL of ice-cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin), followed by incubation on ice for 4 min. Subsequently, 450 μL of ice-cold wash buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20) was added and centrifuged at 600 × g for 6 min. Pellets were resuspended in 25 μL of ATAC-seq transposition mix [12.5 μL 2× Illumina Tagment DNA (TD) buffer; 10.5 μL nuclease-free water; 2.0 μL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit. DNA samples were amplified with PCR ([72 °C; 5 min] and [98 °C; 30 s] followed by 9 cycles of [98 °C; 10 s, 63 °C; 30 s, 72 °C; 1 min] using unique 10-bp dual indexes and NEBNext High-Fidelity 2 × PCR Master Kit (M0541L). Following the first amplification, DNA size selection was performed using solid-phase reversible immobilization (SPRI) beads (AMPure XP [Beckman Coulter, A63881]) at an SPRI to DNA ratio of 0.5. The supernatant was further mixed with SPRI beads at a SPRI to DNA ratio of 1.2. The resulting supernatant was discarded, and the magnet-immobilized SPRI beads were washed twice with 80% ethanol. DNA was subsequently eluted in 20 μL of EB elution buffer (QUIAGEN, included in 28004). The size-selected DNA was amplified with an additional 9-cycle PCR. Subsequently, the amplified DNA was purified with Ampure XP (SPRI to DNA ratio of 1.7) and eluted with 25 μL of buffer EB elution buffer.