GSM4932257: 25_10_TDP_2_S14; Mus musculus; ATAC-seq
To probe chromatin accessibility, we harvested neurons 20, 40 and 60h following transduction of (PR)50, TDP-43, or GFP expressing lentiviruses.  We used direct lysis of plated neurons, followed by isolation of released nuclei. Nuclei were then centrifuged for 10 min at 500 RCF in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was carefully removed with two pipetting steps, as described before, and nuclei were resuspended in 50μl of transposition mix (25μl 2× TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide), 2.5μl transposase (100 nM final), 16.5μl PBS, 0.5μl 1% digitonin, 0.5μl 10% Tween-20, and 5μl water) by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator columns.   The complete eluate underwent PCR, as follows. After initial extension, 5 cycles of pre-amplification using indexed primers (Buenrostro et al., 2015) and NEB Next High-Fidelity 2X PCR Master Mix (NEB) were conducted, before the number of additional cycles was assessed by quantitative PCR using SYBR Green. Typically, 7-9 additional cycles were run. The final library was purified using a MinElute PCR kit (QIAGEN) and quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent).