GSM9309233: Day3 Definitive Endoderm ATAC-seq rep1; Homo sapiens; ATAC-seq
For ATAC-seq, 50,000 cells from replicate biological wells were collected from independent differentiations to the desired stage, and lysed in 50mL of ATAC-lysis buffer (10mM Tris-HCl, pH 7.4. 10mM NaCl, 3mM MgCl2, 0.1% NP-40) to obtain a crude nuclei prep. All centrifugation steps were performed at 4°C at 2000 rpm. The nuclei pellet was then resuspended in the 50ul of the transposition reaction mix (25ul Tagment DNA buffer, 2.5ul TD Tn5 Transposase enzyme, 22.5ul nuclease-free water) (Illumina; 20034197). The transposition reaction was incubated at 37°C for 30 min on a thermomixer with constant gentle shaking at 1000 rpm. Immediately after transposition, DNA was purified using a Qiagen MinElute PCR Purification (Qiagen; 28004 kit) and eluted in 10mL Elution Buffer. The eluted DNA was then amplified in a reaction with 25mL NEBNext High-Fidelity 2x PCR Mastermix (NEB; M0541L) and custom 25um Nextera PCR Primers (Ad1_noMx universal primer, 0.5um Ad2.x indexing primer). PCR was performed as follows: 1 cycle of 72°C for 5min, 98°C for 30s, 5 cycles of 98°C for 10s, 63°C for 30s, 72°C for 1min. 5ul of the amplified DNA was then used to perform qPCR to determine the optimal number of additional cycles to prevent amplification saturation of DNA libraries. In all cases, either 4 or 5 additional cycles of PCR was performed at: 98°C for 10s, 63°C for 30s and 72°C for 1 min. Double size selection of amplified libraries (0.5x – left sided, 1.8x – right sided) was performed using AMPure XP beads and the DNdA was eluted in a final volume of 20ul in 0.1x TE buffer. Purified libraries were sequenced on an Illumina NextSeq 500 instrument with a 2 × 75bp read length. Library construction performed using Nextera XT DNA Library Preparation Kit (FC-131-1024) according to manufacturer's instructions