title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [ATAC]
design: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [ATAC]
library_construction_protocol: 50,000 iPSCs were collected and centrifuged at 500g for 5 minutes in a pre-chilled centrifuge and then briefly resuspended in ice-cold ATAC Resuspension Buffer (ATAC-RSB buffer: Tris-HCl pH 7.4 10mM, NaCl 10mM, MgCl2 3mM) supplemented with NP-40 0.1%, Tween-20 0.1% and Digitonin 0.01%. Samples were incubated on ice for 3 minutes. Lysis was washed out with ATAC-RSB supplemented with Tween-20 0.1% without NP-40 or Digitonin. Nuclei were then centrifuged for 10 minutes at 500g in a pre-chilled centrifuge. To allow transposase reaction, samples were resuspended in 50ul of ice-cold transposition mixture (TD buffer 2X, MEDS-loaded Tn5 100nM, PBS 33%, digitonin 0.01%, Tween-20 0.1%) and then incubated for 30 minutes at 37 C on agitation (1000 rpm). Tn5 was pre-loaded with pre-annealed Mosaic End double-stranded (MEDS) oligonucleotides as described in Picelli et al89.  To clean up the transposase reaction, samples were purified with Zymo DNA Clean and Concentrator-5 kit (Zymo Research), according to manufacturer instructions. Eluted tagmented DNA was PCR amplified for 5 cycles using NEBNext Master Mix (NEB) and barcoded with Unique Dual Indexes (UDIs) which mitigate sample misassignment due to index hopping during de-multiplexing. 5 ul of the total 50 ul PCR reaction were collected for qPCR quantification using Viia7 Real-Time PCR system in order to assess the right number of additional cycles required to obtain optimal complexity during library amplification. 8 final PCR cycles (5 pre-amplification + 3 extra cycles) was established as the gold standard for iPSCs samples, using the following condition: 72°C for 5 min, 98°C for 30s, and thermocycling for 98°C for 10s, 63°C for 30s and 72°C for 1 min.  DNA fragments obtained at the end of library preparation underwent a double-sided size selection to remove primer dimers and fragments larger than 1000bp. To remove DNA fragments > 1000bp, 0.5X volumes of Agencourt AMPure beads XP (Beckman Coulter) were added to the samples, then incubated for 10 minutes at RT. The supernatant, containing DNA fragments < 1000bp, was transferred in a new tube and incubated for 10 minutes at RT with 1.3X original volume AMPure beads. Supernatant, containing primer dimers, was discarded and the DNA-beads complex was washed 3X with Ethanol 80% and eluted in water.