GSM5149228: ATAC Eomes T dKO Day3 rep2; Mus musculus; ATAC-seq
The cells were lysed in 50 μl of cold lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and immediately centrifuged at 500g, 4°C for 10 min. The pellet was resuspended in the transposition reaction mix (25 μl 2x TD buffer, 2.5 μl Tn5 transposase, 22.5 μl H2O) and incubated at 37°C for 30 minutes. After incubation, the reaction was stopped with the addition of PB buffer (Qiagen) and the tagmented DNA was purified using the MinElute kit (Qiagen). The DNA was combined with the ATAC index PCR primers and 2x Kapa HiFi Hotstart Ready Mix and pre-amplified (98°C 30 seconds, 5x [98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute] in a 50 μl volume. To determine the remaining cycles to avoid potential over-amplification, 5 μl of the pre-amplification mix was combined with the primers, 1x Evagreen SYBR green (Jena Biosciences) and 2x Kapa HiFi Hotstart Ready Mix in a 15 μl total volume and run for 30 cycles on a StepOne Plus. The remaining 45 μl of pre-amplified samples were amplified for a further 6-7 cycles and the libraries were purified using MinElute column (Qiagen). The libraries were quantified using the Qubit DNA HS assay and the library sizes were validated using DNA HS Bioanalyzer chips.