GSM2564035: Control rep2 b2; Homo sapiens; ATAC-seq
ATAC-seq was performed as previously described by Buenrostro et al. (PMID: 24097267) with three modifications: (i) To reduce mitochondrial DNA contamination, we substituted 0.1% Ipegal with 0.01% Tween in the nuclear lysis buffer. (ii) Additional tagmentation buffer (TD; 2x) was prepared as follows: 20 mM Tris(hydroxymethyl)aminomethane and 10 mM MgCl2; adjusted to pH 7.6 with 100% acetic acid before addition of 20% (vol/vol) dimethylformamide (Wang et al., 2013). (iii) We performed the PCR amplification in a 25ul total volume rather than 50ul to save half of our tagmented DNA for backup. After 31 days in vitro, iPSC neurons were trypsinised and counted to provide 50,000 live cells for ATAC-seq. iPSC-derived neurons were lysed with 0.01% Tween, 10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and nuclei pelleted, washed and transposed upon addition of 2.5 uL Nextera Tn5 Transposase in 1x TD buffer (Nextera Kit, Illumina) and incubation at 37°C for 30 mins. Immediately following transposition, DNA fragments were purified using a Qiagen MinElute PCR Purification Kit (Qiagen) following the manufacturer’s instructions. Transposed DNA was amplified with dual indexed Nextera PCR primers for 5 cycles prior to removing a 5ul aliquot from each sample to test further amplification requirements by qPCR. Rounding up the cycle threshold from each sample decides how many additional cycles each sample needs to be amplified. For these samples, we needed to re-array and continue amplifying the remaining 20ul for 5-8 cycles. Final libraries were cleaned twice with 1.5X Ampure XP SPRI beads and eluted in 12ul of water.
GEO Accession
GSM2564035