GSM2611673: MEF_before_NT; Mus musculus musculus x M. m. castaneus; ATAC-seq
Cultured cells were harvested, washed with PBS, and then transferred to cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% IGEPAL, 0.5% Triton X-100). Triton X-100 was needed especially for permeabilizing cells after nuclear transfer. Cells in lysis buffer were collected by centrifugation and the cell pellet was resuspended in the transposon reaction mix (25 μl 2x TD Buffer [Illumina, FC-121-1030], 2.5 μl Tn5 Transposes [Illumina, FC-121-1030], 22.5 μl Nuclease Free H2O). A total of 50 μl of the transposon reaction was used for 50,000 cells while 3,000 cells were incubated in 10 μl of the transposon reaction. Transposition reaction was performed at 37oC for 30 min. After the transposition reaction, transposed DNA was purified using a QIAGEN MinElute kit and used for the subsequent PCR amplification. Cultured cells with 1,000 were directly subjected to PCR amplification without using the purification kit. Transposed DNA was amplified by two rounds of PCR using NEB Next High Fidelity Master Mix (New England Labs, M0541) and the Customized Nextera PCR primers totaling 50 μl (Buenrostro et al., 2013). The first PCR cycles varied from 5 to 15 cycles depending on the starting cell numbers. The number of second PCR cycles was determined by qPCR using the 10% of the total PCR reaction in order to amplify the DNA in the exponential growth phase. The amplified DNA was purified using QIAGEN PCR Cleanup kit and finally resuspended in 20 μl of elution buffer. The quality of library was checked on polyacrylamide gels and quantified using the KAPA library quantification kit for Illumina sequencing platforms (KAPA Biosystems).
GEO Accession
GSM2611673