title: Illumina HiSeq 2000 sequencing; RNA-seq of H3.3K4A/K36A mutant ESCs and neurons
design: RNA-seq of H3.3K4A/K36A mutant ESCs and neurons
library_construction_protocol: 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).    RNAs were extracted from 1 Mio. cells using RNeasy Kit (Qiagen), followed by DNase digestion using TURBO DNase (Thermo Fisher).  RNA was quantified using Nanodrop and RIN score quality assessment was determine using Bioanalyzer. PolyA-mRNA was isolated from 1 microg of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) before sequencing library preparation. mRNAs were isolated from 1 microg of total RNA using a PolyA selection kit (NEB) and sequencing libraries were prepared following instructions from NEBs Ultra Library Preparation Kit for Illumina. All samples were barcoded, amplified in 12x PCR cycles, pooled and sequenced on a HiSeq2500 Sequencer (Illumina) using a 50 bp single-end run.
attributes.tag: Experimental Factor: time
attributes.value: 12
attributes.tag: Experimental Factor: genotype
attributes.value: H3f3a -/-