GSM4823255: Drosophila S2R+ cells - 25°C_ATAC-seq_Rep1; Drosophila melanogaster; ATAC-seq
Cell harvest was performed quickly using 0.025% Trypsin-EDTA (ThermoFisher Scientific, 25200-056) within the incubators and at the temperatures used during the preceding 24 hour incubation, with solutions pre-equilibrated to the different incubation temperatures. The culture medium was removed and cells were rinsed once with PBS before addition of 1 mL Trypsin-EDTA solution for 5 minutes. The released cells were transferred into a 15 mL Falcon tube and combined with four rinses of the plates (each with 1 mL of complete medium). The resulting master cell suspension was diluted into ice-cold complete medium in order to obtain an Eppendorf tube with 75000 cells in 1 mL for each aliquot. Cells were sedimented (500×g for 5 min, 4°C) and washed once with 50 µL PBS. Cells were resuspended and lysed with 50 µL of cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40). Lysed cells were sedimented (500×g for 10 min, 4°C). Tagmentation was performed during 30 minutes at 25°C in a 50 µL reaction in TD Buffer (Illumina, FC-121-1030) containing 2.5 µL Tn5 Transposes (Illumina, FC-121-1030) (Buenrostro et al. 2013). Tagmented DNA was purified using a Qiagen MinElute Kit and amplified as described (Buenrostro et al. 2013) using a Nextera forward primer (Ad1_noMX) in combination with a replicate-specific bar-coded reverse Netera index primer (Ad2.1 to Ad2.9). Libraries were purified using Qiagen PCR Cleanup Kit.