GSM4648278: ATAC_WT_REP2; Mus musculus; ATAC-seq
For each cell line, three independent RNA samples were isolated at different time points. 50,000 cells were sorted into 500 μL PBS and immediately processed as described under "library construction protocol" To prepare the ATAC sequencing libraries, 50,000 cells were sorted into 500 μL PBS. The libraries were prepared as previously described (Buenrostro et al., 2013) with slight modifications (Lara-Astiaso et al., 2014). Briefly, the cells were lysed with 25 μL cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM MgCl2, 0.1% Igepal CA-630) and the nuclei were pelleted by centrifugation for 25 min at 4°C and 500 g in a swing rotor with low acceleration and brake settings. The pellet was resuspended in 25 μL reaction mix containing 2 μL Tn5 transposase and 12.5 μL TD buffer (Nextera DNA library preparation kit, Illumina), and incubated at 37°C for 1 h. Next, 5 μL cleanup buffer (900 mM NaCl, 30 mM EDTA) with 2 μL 5% SDS and 2 μL Proteinase K (NEB) were added, and the samples were incubated at 40°C for 30 min. Subsequently, the DNA was purified using AMPure XP beads (Beckman Coulter) and PCR-amplified with Kapa HiFi HotStart ReadyMix (Kapa Biosystems) and indexing primers published previously (Buenrostro et al., 2013). Then, the DNA library fragments were selected for <600-bp fragments and purified using AMPure XP beads. The concentration and fragment size of the final libraries were measured using a Qubit fluorometer (Life Technologies) and TapeStation (Agilent Technologies). The samples were sequenced with an average of 25 million reads per sample using a NextSeq 500 (Illumina).
GEO Accession
GSM4648278