title: H9_128d_org1
organism: Homo sapiens
description: Protocols: Single cell suspensions from organoids and earlier stages of organoids development (EB stage until neuroepithelium stage) were obtained by Papain based dissociation (Miltenyi Biotec, Neural tissue dissociation kit (P)) using microdissected regions or organoids or whole organoids. Induced pluripotent stem cell single cell suspension were obtained by detaching stem cells from matrigel coated culture dishes using TrypLExpress, followed by multiple rounds of washing the cells. Single cells were encapsulated along with beads and reverse transcription reagents into droplets using 10X microfluidic chip system. Reverse transcription of first strand cDNA was performed in droplets. JoC and SandraA were reprogrammed according to Okita et al. (PMID 23193063) from PBMCs, 409b2 from fibroblasts according to Okita et al. (PMID 21460823). HipSci cell lines were reprogrammed using Sendai virus. PR818-5 (Marchetto et al. 24153179) and SC102A1 were reprogrammed using retroviral vectors. Stem cells were cultured under standard feeder-free conditions. The stages EB, neuroectoderm and neuroepithelial are stages during organoid differentiation. Induced pluripotent stem cells were maintained in feeder-free culture on matrigel coated plates. Cerebral organoids were generated according to the protocol by Lancaster et al. (Nature Protocols, 2014). Single stranded cDNA was cleaned up and amplified after breaking the droplets generated during the sample collection using the 10X 3' Single cell Kit v2 according to the manufacturer's instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay. Libraries were constructed following the 10X library construction protocol v2. Briefly, cDNA was fragmented, end repaired and adaptor sequences were ligated, followed by a PCR to itroduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-04T04:04:47Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-08-07T13:26:36Z
attributes.tag: External Id
attributes.value: SAMEA5857812
attributes.tag: INSDC center name
attributes.value: MPI-EVA
attributes.tag: INSDC first public
attributes.value: 2019-10-04T04:04:47Z
attributes.tag: INSDC last update
attributes.value: 2019-08-07T13:26:36Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7552_2:H9_128d_org1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: cerebral organoid
attributes.tag: individual
attributes.value: H9
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: phenotype
attributes.value: 4 month
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-7552_2:H9_128d_org1
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female