title: GSM4929444: Bap1flfl_ATAC.Input_UNT_rep2; Drosophila melanogaster; Mus musculus; ATAC-seq
library_construction_protocol: For cATAC-seq, 1×107 ESCs (untreated and OHT-treated) were mixed with 4×106 Drosophila SG4 cells in 1x PBS and then lysed in 1 ml HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05% NP40 (IGEPAL CA630), 1 mM PMSF, 3 mM DTT, 1xPIC (Roche)) for 1 min at room temperature. Nuclei were recovered by centrifugation at 1000 g for 5 min at 4°C and washed three times in ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2). The concentration and integrity of nuclei were assessed using 0.4% Trypan Blue staining (ThermoScientific). Next, 5×105 nuclei were resuspended in Tn5 reaction buffer (10 mM TAPS, 5 mM MgCl2, 10% dimethylformamide) and incubated with Tn5 transposase (25 µM, generated in house as previously described (King and Klose 2017)) for 30 min at 37°C. Tagmented DNA was purified using MinElute columns (QIAGEN) and eluted in 10 μl elution buffer. To control for the Tn5 transposase sequence bias and to determine the exact spike-in ratio for each individual sample, 50 ng of genomic DNA, isolated from the same nuclei preparation by phenol-chloroform extraction, was tagmented with Tn5 transposase (25 µM) for 30 min at 55°C and purified using MinElute columns (QIAGEN). Libraries for cATAC-seq and gDNA-seq were prepared by PCR amplification using the NEBNext High-Fidelity 2X PCR Master Mix and custom-made Illumina barcodes (Buenrostro et al. 2015). Libraries were purified by two rounds of Agencourt AMPure XP bead cleanup (1.5× bead:sample ratio). The average size and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced using the Illumina NextSeq 500 platform in biological quadruplicate using 80 bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM4929444