GSM8247173: 3f2_sgrna2_gata4; Mus musculus; ATAC-seq
ATAC-seq during reprogramming was carried out as previously described with minor modifications (Buenrostro, et al., 2015). ATAC-seq libraries were prepared using NEBNext High-Fidelity 2X PCR Master Mix (NEB, #M0541), a uniquely barcoded primer per sample, and a universal primer. Samples were first subjected to 5 cycles of initial amplification. To determine the suitable number of cycles required for the second round of PCR (to minimize PCR bias) the library was assessed by quantitative PCR. Briefly, a 5 μL aliquot of the initial amplification sample was used for 20 cycles of qPCR. Linear Rn versus cycle was plotted to determine cycle number corresponding to 1/3 of maximum fluorescent intensity. For each sample, the remaining 45 μL of initial tagmented PCR product was further amplified for 5 more cycles using Nextera primers. Samples were subject to a dual size selection (0.55x–1.5x) using SPRIselect beads (Beckman Coulter,B23317). Fragment distribution of libraries was assessed with an Agilent Bioanalyzer and finally, the ATAC libraries were sequenced on an an Illumina NovaSeq 6000 (PE-50, 20 million reads per sample).