organism: Homo sapiens
description: Protocols: Total RNAs were isolated from either hMLOs, 2D-DA neurons, or undifferentiated hESCs using TRIzol Reagent (Invitrogen). DNA contaminants were digested with DNASE I (Ambion). DA neuron differentiation was performed follow floor-plate based neural induction protocol (Kriks et al., 2011). The hESCs were plated at 35 x 103 cells per cm2 and grown on matrigel in Basal medium 1 containing DMEM, 15% KSR, 2mM L-Glutamine and 10μM β-mercaptoethanol, with addition of 100nM LDN193189 (Stemgent) and 10μM SB431542 for 24 h. On day 1, medium was changed to Basal Medium 1 with addition of 100nM LDN193189, 10μM SB431542, 100ng/ml SHH-C25II, 2μM Purmorphamine (Stemgent) and 100ng/ml FGF8 for 2 days. On day 3, medium was changed with addition of 3μM CHIR99021. By day 5, medium was gradually shifted to N2 medium containing DMEM, N2 supplement, 2mM L-Glutamine and 10μM β-mercaptoethanol (Basal Medium 2) for 2 days interval as described previously (Chambers et al., 2009). On day 11 onwards, Basal medium 3 containing Neurobasal/B27/L-Glutamine (Invitrogen) was changed supplemented with 3μM CHIR99021 for 2 days. After which, Basal medium 3 supplemented with 10μM DAPT (Sigma-Aldrich), 0.2mM Ascorbic acid, 20ng/ml BDNF, 20ng/ml GDNF, 0.2mM db-cAMP and 1ng/ml TGF-β3 (Invitrogen) was used until day 20, media were changed every other day. On day 20, cells were dissociated using 0.05% Trypsin-EDTA and replated under high cell density (300 - 400 x 103 cells per cm2) on dishes pre-coated with 0.1mg/ml poly-lysine and 1μg/ml Laminin. Cells were grown in Neurobasal/B27 medium containing 10μM DAPT, 0.2mM Ascorbic acid, 20ng/ml BDNF, 20ng/ml GDNF, 0.2mM db-cAMP and 1ng/ml TGF-β3, and changed fresh medium every other day until experimental analysis as indicated. For RNA-seq library preparation of hMLOs and 2D-DA neurons, the total RNA was further purified by column (Puelink RNA Mini kit, Ambion), and was prepared using 4μg of total RNA according to manufacturer's protocol (TruSeq RNA Sample Preparation Kit v2, Illumina). Samples were multiplexed and sequenced single read 150bp paired end (HiSeq 2000, Illumina).
attributes.tag: ENA first public
attributes.value: 2016-08-24
attributes.tag: ENA last update
attributes.value: 2016-06-27
attributes.tag: External Id
attributes.value: SAMEA4051971
attributes.tag: INSDC center alias
attributes.value: GIS
attributes.tag: INSDC center name
attributes.value: Genome Institute of Singapore
attributes.tag: INSDC first public
attributes.value: 2016-08-24T17:08:01Z
attributes.tag: INSDC last update
attributes.value: 2016-06-27T18:04:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4868:GIS_2D_DA_neuron_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: cell
attributes.tag: sample name
attributes.value: E-MTAB-4868:GIS_2D_DA_neuron_1