Illumina HiSeq 2000 sequencing; Sox2 ChIP-seq in mESCs and neurons
Sox2 ChIP-seq in mESCs and 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).     For each replicate 24-34 million cells were harvested and cross linked for 15 minutes in 1.5% paraformaldehyde in PBS at room temperature. Cross-linking solution was quenched with 125 mM Glycine  for 5 min. Fixed cells were spun down and washed twice with ice cold PBS freshly supplemented with protease inhibitors. Cross-linked pellets were snap frozen in liquid nitrogen and kept at -80 C. Chromatin was extracted by cell lysis and centrifugation and sonicated with 5 cycles in a cooled Bioruptor at 4C.Samples were spun down to separate insoluble chromatin. Soluble fragmented chromatin was used as ChIP input. Both soluble input and precipitated pellet were resolved on an agarose DNA gel to check the digestion pattern across replicates and samples. 5% of each chromatin inputs were set aside before the IP. Samples were eluted from ProteinG Dynabeads using SDS-elution buffer (50 mM Tris-HCl ph8.0, 10 mM EDTA, 1% SDS) at 65C for 30 minutes, shaking at 1500 rpm.  Proteinase K was added (0.2 mg/ml final) to eluted samples and digestion was carried out for 2 hours at 55C followed by PCR purification (Qiagen). For ChIP Input 100 ng of prepared sonicated DNA was used for library preparation. For ChIP, entire IP-eluate was used for library preparation.  Sequencing libraries were prepared using DNA Ultra II library preparation kit (NEB) according to the manufacturer instructions.  Number of Amplification cycles was adjusted depending on ChIP (5-7 cycles). Samples were barcoded, pooled and sequenced on Illumina HiSeq2000 Sequencer (50 bp single-end mode).