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). hPSC lines before passage 40 were used to generate hMLOs. The hPSCs were dissociated from intact colonies to single cells with TrypLETM Express (Life Technologies), and 10,000 cells were plated in each well of low-cell-adhesion 96-well culture plates with V-bottomed conical wells (Sumitomo Bakelite) to form uniform EBs. The wells contained neuronal induction medium containing DMEM/F12:Neurobasal (1:1), 1:100 N2 supplement (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media-nonessential amino acid (Invitrogen), and 0.1% β-mercaptoethanol (Invitrogen) supplemented with 1μg/ml heparin (Sigma-Aldrich), 10μM SB431542 (Stemgent), 200 ng/ml Noggin (Prospec), 0.8μM CHIR99021 (Reagents Direct), and 10μM ROCK inhibitor Y27632 (Calbiochem). The ROCK inhibitor was added for the first 48 h, and the neuronal induction medium was changed on day 2. On day 4, hMLOs were cultured with the addition of 100 ng/ml SHH-C25II (R&D Systems) and 100 ng/ml FGF8 (R&D Systems) for midbrain patterning. After 3 days, the hMLOs started to extrude neuroectodermal buds. At this point, the media was completely removed, and 30μl of reduced growth factor Matrigel was immediately added to each well using a pipettor equipped with a pre-chilled 200μl pipette tip. The Matrigel-embedded midbrain organoid was placed into a 37°C incubator for 30 min to allow the Matrigel to solidify and was grown in tissue growth induction medium containing Neurobasal medium, 1:100 N2 supplement (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media-nonessential amino acid (Invitrogen), and 0.1% β-mercaptoethanol (Invitrogen) supplemented with 2.5 μg/ml insulin (Sigma-Aldrich), 200 ng/ml laminin (Sigma-Aldrich), 100 ng/ml SHH-C25II (R&D Systems), and 100 ng/ml FGF8 (R&D Systems) for 24 h. Once the hMLOs were embedded in Matrigel, a more expanded neuroepithelium began to form in the organoids. To promote growth and differentiation, the hMLOs were transferred into ultra-low-attachment 6-well-plates (Costar) by pipetting using a cut 1000 μl pipette tip. The plates contained the final differentiation media, which consisted of Neurobasal medium, 1:100 N2 supplement (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media-nonessential amino acid (Invitrogen), 0.1% β-mercaptoethanol (Invitrogen), 10ng/ml BDNF (Peprotech), 10 ng/ml GDNF (Peprotech), 100 μM ascorbic acid (Sigma-Aldrich), and 125 μM db-cAMP (Sigma-Aldrich). The hMLOs were cultured using an orbital shaker to enhance nutrients and oxygen exchange (Lancaster and Knoblich, 2014). Antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) were included in the culture media to prevent potential bacterial contamination over long-term culture. The medium was replaced every 3 days. 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: SAMEA4051978
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_hMLO_5
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_hMLO_5