GSM8636058: rtf1_AAAA_bio3; Mus musculus; ATAC-seq
Nuclei were resuspended in 50 µL Transposase mix (25 µL 2x Tagmentation Buffer, 16.5µL 1x PBS, 0.5 µL 10% Tween-20, 0.5 µL 1 % Digitonin, 2.5 µL Tagmentase, 5 µL nuclease-free water) and incubated at 37°C for 30 min in a thermomixer shaking at 1000 rpm. Immediately, DNA was purified using the Zymo DNA Clean and Concentrator Kit per manufacturer guidelines and eluted in 10 µL water. DNA fragments were PCR amplified (2.5 µL 25µM Nextera i5 and i7 primers, 10 µL water, 25 mL NEBNEXT High-Fidelity 2x PCR Master Mix, and 10 µL DNA). Initial amplification occurred for 5 cycles with conditions: 72°C 5 min, 98°C 30 sec, and 5 cycles (98°C 10 sec and 63°C 30 sec) and 72°C 1 min elongation. Quantitative PCR was performed to determine the number of additional cycles required (1µL library DNA, 1 µL NXT PCR primers at 2 µM, 3 µL nuclease-free water, 5 µL 2x KAPA SYBR green, and 9 µL qPCR Master Mix). qPCR conditions were 98°C 30 sec followed by 20 cycles (98°C 10 sec, 63°C 30 sec, 72°C 1 min). Remaining library was PCR amplified for 4 cycles. Amplified libraries were run on a 1.5% agarose gel and fragments sized 150-700 bp were then gel-extracted. Libraries were paired-end sequenced on a Illumina NextSeq2000 to a depth of ~50 million uniquely mapped reads per sample.