GSM5800027: B6xMOLF-ATAC-10m-B1; Mus musculus; ATAC-seq
GSM5800027
MEFs from 6-well dish were washed twice in 1 mL cold PBS and pelleted each time by spinning at 300 g for 5 min at 4oC. 50,000 MEFs were resuspended in 50 uL cold ATAC lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 0.1% Tween 20, 0.01% digitonin) and incubated for 3 min on ice.  Lysed cells were washed once in 1 mL ATAC wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20) by gently inverting the tube 3 times and pelleted by spinning at 500 g for 10 min at 4oC. Pelleted nuclei were resuspended in 50 uL transposition mix (25 uL 2x TD Buffer (Illumina (San Diego, CA, USA) 20034197), 2.5 uL TDE1 transposase (Illumina 20034197), 0.5 uL 10% Tween 20, 0.5% 1% digitonin, 16.5 uL PBS, 5 uL NF-H2O) and incubated for 30 min at 37oC with a Thermomixer set to 1,000 rpm. Samples were purified with MinElute PCR Purification Kit (QIAGEN 28004) per manufacturer's instructions and eluted in 13 uL NF-H2O. Libraries were amplified by adding the following to 10 uL purified DNA: 2.5 uL 25 uM Ad1 universal primer, 2.5 uL 25 uM Ad2.* indexing primer, 25 uL NEBNext Hi-Fi 2x PCR Master Mix (NEB (Ipswich, MA, USA) M0541S), 10 uL NF-H2O. After an initial 5 PCR cycles, libraries were quantified by qPCR by adding the following to 5 uL partially amplified DNA: 0.5 uL 25 uM Ad1 universal primer, 0.5 uL 25 uM Ad2.* indexing primer, 5 uL NEBNext Hi-Fi 2x PCR Master Mix, 0.15 uL 1x SYBR Green I (Thermo Fisher S7563), 3.85 uL NF-H2O. All primer sequences referenced are described in Buenrostro et al., 2015. The number of additional PCR cycles required for amplifying remaining libraries was determined by number of qPCR cycles needed to reach 1/3 of the maximum SYBR green signal. Libraries were purified with AMPure XP beads (0.5x volume; Beckman Coulter (Indianapolis, IN, USA) A63881) and the supernatant was retained to remove large fragments. Primer dimers were removed by a subsequent cleanup with AMPure XP beads (1.3x initial volume) and libraries were eluted in 20 uL NF-H2O. Libraries were sequenced on an Illumina NextSeq 500 with 40 bp paired-end reads.