title: mRNA_H3.3K36A-3_D12
organism: Mus musculus
description: Protocols: 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: ENA-FIRST-PUBLIC
attributes.value: 2019-04-11T04:02:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-06-15T10:50:11Z
attributes.tag: External Id
attributes.value: SAMEA4730298
attributes.tag: INSDC center name
attributes.value: EMBL
attributes.tag: INSDC first public
attributes.value: 2019-04-11T04:02:20Z
attributes.tag: INSDC last update
attributes.value: 2018-06-15T10:50:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6821_2:mRNA_H3.3K36A-3_D12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 129-B13
attributes.tag: cell type
attributes.value: neuron
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genetic modification
attributes.value: induced mutation
attributes.tag: genotype
attributes.value: H3f3a -/-; H3f3b K36A
attributes.tag: sample name
attributes.value: E-MTAB-6821_2:mRNA_H3.3K36A-3_D12
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: time
attributes.value: 12