GSM6422262: AW4, wildtype, ATAC-seq; Mus musculus; ATAC-seq
Neurospheres were passaged using 1X TrypLE Express (Thermofisher cat#:12604013) with gentle trituration and passed through a 40µm cell strainer. For ATAC-seq library preparation, we utilized the Omni-ATAC-seq protocol first described by (Buenrostro et al., 2013, 2015), with minor changes. For each biological replicate, 50,000 cells were counted and washed in ice-cold 1ml 1X PBS. To isolate nuclei, the cells were pelleted and resuspended in 50μl of ice cold Lysis Buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and gently mixed by pipet 3 times to resuspend. Cells were incubated on ice in Lysis Buffer for 3 minutes before being washed with 1ml Wash Buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20) 3 times to stop the lysis reaction. Nuclei were spun down at 500G for 10 minutes and the supernatant was discarded. For each sample, a Transposition Reaction Mix was made using the Nextera DNA Library Prep Kit (25μl 2X TD Buffer (Tagment DNA Buffer), 16.5μl 1X PBS, 0.5μl 10% Tween-20, 0.5μl 1% Digitonin, 2.5μl Tn5 Transposase (Tagment DNA Enzyme 1), 5μl nuclease-free H2O. Each nuclear pellet was resuspended in the Transposition Reaction Mix and incubated at 37°C for 30 minutes in a heat block with occasional mixing. DNA was purified using a Qiagen MinElute Reaction Cleanup Kit and eluted in 10μl of 10mM Tris-HCl pH 8.0. The isolated DNA was used as the input into the PCR amplification/ Library Generation step. Each PCR reaction consisted of 10μl of purified transposed DNA, 10μl nuclease-free H2O, 2.5μl of Ad1_noMx forward primer, and 2.5μl of the reverse primer. Samples we PCR amplified using the following settings: 72°C for 5 minutes, then 98°C for 30 seconds followed by 16 cycles of (98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minute) then 72°C for 5 minutes and hold at 4°C. The number of PCR cycles to use was determined by using qPCR on 1/5th of the library from each sample. After the first 5 cycles above, the libraries were removed and 5μl of the PCR reaction was subjected to qPCR for 20 cycles. The number of cycles needed to reach a third of the maximum fluorescence for each sample was used as the remaining number of cycles to add, in this case, 11 cycles, for a total of 16 cycles. Libraries were purified using AMPure XP beads to remove primer dimers using the standard protocol. To remove the small fragments, 1.8X bead volume was added to each library. For removing large fragments >1000bp, 0.5X bead volume was used on the eluate obtained above. Once finished, the purified libraries were quality controlled using Agilent High Sensitivity DNA bioanalysis chip to look for nucleosome periodicity and measured using QuBit 2.0 for DNA concentration, both following manufacturer's instructions. Samples were sequenced using 50bp paired-end reading at roughly 50 million reads per sample.