NextSeq 500 paired end sequencing; ATAC-seq of mESCs differentiation into neurons
ATAC-seq of mESCs differentiation into neurons
mESC growth:  Mouse embryonic stem cells (129B-13) were cultured in ESC media containing Knockout-DMEM (Thermo Fisher) with 15% EmbryoMax FBS (Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression Facility at EMBL Heidelberg), 1% non-essential amino acids, 1% Glutamax, 1% Pen/Strep, 1% of 55mM beta-Mercaptoethanol solution. Cells were maintained at 37C with 5% CO2. mESCs were used as neuronal differentiation Day 0 (D0).    Neuronal Differentiation:  mESCs were differentiated into glutamatergic neurons according to Bibel et al. (Nature Protocols, 2007) with small modifications. mESCs were resuspended in differentiation media without LIF and grown in suspension using non-adherent plates to promote the formation of embryoid bodies. Differentiation media was changed every two days. On day 4 retinoic acid was added to the media. Embryoid bodies were cultured for 4 additional days in the presence of retinoic acid to obtain neural precursor cells on differentiation day 8. For neural monolayer culture, plates were pre-coated with Poly-D-Lysin and Laminin. On day 8, Embryoid bodies were dissociated with trypsin and cells were plated in N2 media (regular DMEM supplemented with 1xN2 and 1xB27-VitaminA (Thermo Fisher)) at a density of 200.000 cells/cm2 on precoated plates. N2 media was changed every two days without exposing neuron culture to oxygen.   Four days after plating, neurons were harvested after a total of 12 differentiation days (D12).    20.000 cells were harvested and washed once in cold PBS for 5 min at 500 x g at 4 C. The pellet was gently resuspended in 50ul of cold lysis buffer and spun down immediately at 500 x g for 10 min at 4 C. Transposition reaction mix with the enzyme and transposition buffer from Illumina Nextera DNA Library Preparation Kit was added to the pellet and incubated for 30 min at 37 C. The DNA was purified after transposition using a Qiagen MinElute kit and eluted in 10ul of the provided elution buffer. Eluted after transposition DNA was PCR amplified for a total of 11-13 cycles using barcoded primers from Illumina Nextera XT Index Kit v2. DNA was purified and adapters were removed using Ampure beads (1.4:1.0 beads:sample ratio). The quality and concentration of the eluted libraries were determined using an Agilent Bioanalyzer HS chip and a Qubit fluorometer. Libraries were sequenced on Illumina NextSeq 500 in paired-end mode.