GSM9536338: ATAC-seq 20201013_nih3t3_cas9_empty_vector_rep3; Mus musculus; ATAC-seq
Cells were collected and washed with ice-cold PBS. Cell pellets were resuspended in ATAC-seq RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% IGEPAL-630, 0.1% Tween-20, and 0.01% digitonin, and incubated on ice for 10 minutes. ATAC-seq RSB containing 0.1% Tween-20 (without IGEPAL-630 or digitonin) was then added for washing. Nuclei were pelleted in a pre-chilled centrifuge. Nuclei were incubated with Tn5 transposase and tagmentation buffer at 37°C for 30 minutes. Stop buffer and Proteinase K were added, and samples were incubated at 55°C for 30 minutes to terminate tagmentation and remove proteins. PCR was performed to amplify the ATAC library for 5 cycles using KAPA HiFi PCR kit. To determine additional amplification cycles for each sample, qPCR was performed, and the additional cycle number was determined as the cycle reaching one-third of maximal abundance in the qPCR amplification curve. All samples were then amplified for another N+3 cycles, followed by final extension. Libraries were purified with DNA clean beads and sequenced as paired-end libraries on Illumina platforms.