title: GSM8127419: mESC, Fix, rep1; Mus musculus; ATAC-seq
library_construction_protocol: To permeabilize cells, the cell pellets were resuspended in 50 μl of ATAC-seq RSB containing 0.1% IGEPAL-630, 0.1% Tween-20, and 0.01% digitonin and incubated on ice for 10 min. Afterwards, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the samples mixed by inverting the containing tubes for six times.     Nuclei were then centrifuged for 10 min at 500 r.c.f. in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was removed, as for Native group, nuclei were incubated with Tn5 transposome and tagmentation buffer at 37°C for 30min (Novaprotein, N248). As for Fix and 1,6-Hex+Fix conditions, nuclei were then incubated with Tn5 transposome and tagmentation buffer at 55°C for 30min. After tagmentation, stop buffer was added directly into the reaction. For the native condition, samples were incubated at 55°C for 30min to terminate the tagmentation.  The Fix and 1,6-Hex+Fix samples were incubated at 55°C overnight to terminate the tagmentation and to reverse crosslink the samples. PCR was performed to amplify the library for 5 cycles using KAPA HiFi PCR Kit (Kapa Biosystems, kk2102)  with the following PCR conditions: 72°C for 5min; 98°C for 3mins; and thermocycling at 98°C for 20s, 63°C for 30s. The determine the optimal amplification for each sample, 5ul PCR mix was diluted with 5ul H2O (Thermo Scientific, 10977015) and combined with 2x SYBR qPCR supermix (Novoprotein, E096) to perform qPCR with following conditions: 98°C for 3mins; and thermocycling for 30 cycles at 98°C for 20s, 63°C for 30s and 72°C for 1mins. The additional number of PCR rounds (N) was determined as the qPCR cycle that reached the 1/3 maximum abundance on qPCR amplification curve. All samples were amplified for another N+3 cycles with the following PCR conditions: 98°C for 3mins; thermocycling at 98°C for 20s, 63°C for 30s and 72°C for 3min; and 72°C for 5mins for final extension. The library was sequenced on Illumina HiSeq X Ten with an average depth of 17.5 million reads per library.