title: GSM8826688: Run01_Repli-ATAC_4h_noIP; Homo sapiens; ATAC-seq
library_name: GSM8826688
library_construction_protocol: For samples labeled with 50 µM BrdU or nocodazole, cells were lysed via incubation in SDS-PK buffer (0.5% wt/vol) sodium dodecyl sulfate (SDS), 50mM Tris-HCl, 0.01M EDTA, 1M NaCl and 0.2mg/ml proteinase K) at 56°C for 2h. Genomic DNA was isolated using phenol-chloroform extraction and isopropanol precipitation. For samples labeled with 500 µM BrdU, genomic DNA was isolated using the Puregene Cell Kit (8 x 10^8) (Qiagen, 158043) or Monarch® Genomic DNA Purification Kit (NEB, T3010S) according to the manufacturer's instructions. For Repli-ATAC samples: Collected cells were resuspended in 1 mL ice-cold DPBS buffer and 100K cells were transferred to and pelleted in a 1.5 mL Eppendorf tube at 4°C. The pellet was resuspended in 50 uL cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Nuclei were spun down immediately at 500 x g for 10 minutes at 4°C, supernatant was discarded, and the pellet was stored on ice.  To initiate the transposition reaction, the nuclei were resuspended in transposition reaction mix: 50 µL TD (2x reaction buffer, Illumina, FC-121-1030), 5 µL TDE1 (Nextera Tn5 Transposase, Illumina, FC-121-1030), and 45 µL nuclease-free water (Invitrogen, 10977-015). The transposition reaction was incubated on a thermomixer at 37°C for 30 minutes. Then, tagmented DNA was purified using the MinElute PCR Purification kit (Qiagen, 28004). To amplify transposed DNA fragments from each sample, the following was combined in a 0.2 mL PCR tube: 20 µL transposed DNA, 5 µL nuclease-free water, 25 µL NEBNext High-Fidelity 2x PCR Master Mix (NEB, M0541). Samples were placed in a thermal cycler for 1 cycle of 72°C for 5 minutes to allow for extension of both ends of the primer after transposition. DNA was purified using the MinElute PCR Purification kit.  10 µL of each sample was used for BrdU immunoprecipitation (IP) and 10 µL was stored aside at 4°C as a non-IP control. To denature the DNA in the IP sample, 490 µL of TE buffer was added and the sample was incubated at 95°C for 5 minutes on a thermomixer before cooling in an ice bath. The denatured DNA was collected and added to 60 µL of 10x IP buffer (28.5 mL nuclease-free water, 5 mL of 1 M sodium phosphate, pH 7.0, 14 mL of 5 M NaCl, and 2.5 mL of 10% wt/vol Triton X-100). 40 µL of 12.5 µg/mL anti-BrdU antibody (BD Biosciences, 555627) was added, and the sample was incubated for 1 hour at room temperature with constant rotation. The sample was centrifuged at 17,000 x g for 5 minutes at 4°C and the supernatant was completely removed. 750 µL of ice-cold 1x IP buffer was added before centrifuging and removing the supernatant again. The pellet was then resuspended in 200 µL digestion buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS) with 0.25 mg/mL Proteinase K and incubated overnight at 37°C, then for 60 minutes at 56°C. Next, the sample was purified using AMPure XP Beads (Beckman Coulter, A63881) (2x bead volume) and resuspended in 20 µL Elution Buffer. To amplify the transposed DNA fragments, the following was combined in a 0.2 mL PCR tube: 20 µL transposed DNA, 2.5 µL 25 µM Custom Nextera PCR Primer 1, 2.5 µL 25 µM Custom Nextera PCR Primer 2, 25 µL NEBNext High-Fidelity 2x PCR Master Mix. The following PCR conditions were used: 1 cycle of 72°C for 5 minutes, 98°C for 30 seconds, followed by 5 cycles of 98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minute. The PCR reaction was monitored using qPCR to stop amplification before saturation, in order to reduce GC and size bias61. Finally, the amplified library was purified using the MinElute PCR Purification kit.