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Sample GSM3398392 Query DataSets for GSM3398392
Status Public on Feb 15, 2019
Title RNA-Organoid-D45_53BP1KO3 replicate 1
Sample type SRA
 
Source name Cell Line
Organism Homo sapiens
Characteristics cell type: Human Organoid D45
cell line: derived from H9 (day 45)
genotype/variation: 53BP1 KO
Treatment protocol The EB formation medium (DMEM/F12 with 20% knockout serum replacement, 3% ESC-quality FBS, 2 mM GlutaMAX, 0.1 mM nonessential amino acides,) was supplemented with Dorsomorphin (2 µM), WNT inhibitor (IWR1, 3 µM), TGF-ß inhibitor (SB431542, 5 µM) and Rho Kinase inhibitor (Y-27623, 20 µM). Starting form Day4, EBs were fed with cortical differentiation medium (Glasgow-MEM, 20% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM ß-ME, 1% anti-anti), supplement with WNT inhibitor (IWR1, 3 µM), TGF-ß inhibitor (SB431542, 5 µM), Cyclopamine (2.5 µM) and Rho Kinase inhibitor (Y-27623, 20 µM). On Day17, EBs were embedded in matri-gel droplets and transferred onto low-attachemnt 6-wells and cultured in suspension using DMEM/F-12 supplemented with 1% N2 supplement, 1% Lipid concentrate, 2% B27suppmenent without Vitamin A, and 1% anti-anti under 40% O2/5% CO2 conditions on shaker. Starting from Day30, medium was changed to 50% DMED/F-12, 50% Neurobasal media, 0.5% N2 supplement, 1% GlutaMax, 0.05 mM NEAA, 0.025% human insulin, 0.1 mM ß-ME and 1% anti-anti, supplemented with 2% B27.
Growth protocol Cerebral organoids were generated based on previously published methods with minor modifications20,21. In brief, hESCs were dissociated to single cell suspension using Accutase and seeded onto low-attachment 96-well plates (Costar, 7007) at a density of 9,000 cells per well to aggregate into embryoid bodies (EBs).
Extracted molecule total RNA
Extraction protocol RNA was purified with the GeneJET RNA purification kit (ThermoFisher Scientific, K0732). The cDNA was reverse transcribed from 300 ng of RNA, using SuperScript IV reverse transcriptase (ThermoFisher Scientific, #18090050) with random hexamers. RT reactions were performed in triplicate to minimize variability.
total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s directions (Illumina, San Diego, Ca.) Paired end 100 cycle sequencing was performed on HiSeq 2000 or HiSeq 4000 sequencers according to the manufacturer’s directions (Illumina.)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description RNA-Organoid-D45.all.counts.txt.gz
Data processing Basecalls performed using CASAVA
Paired-end 100-cycle sequencing was performed on NovaSeq 6000 sequencers (Illumina). RNA-sequencing was mapped as described previously (Downing et al., 2012), and HTSeq (version 0.6.1p1) (Anders et al., 2015) was used to obtain gene-level counts and estimated CPM based on GENCODE (v24) (Harrow et al., 2012).
Genome_build: hg19(GRCh37)
Supplementary_files_format_and_content: raw reads count by HTSEQ
 
Submission date Sep 21, 2018
Last update date Feb 15, 2019
Contact name Beisi Xu
E-mail(s) beisi.xu@stjude.org
Organization name St Jude Children's Research Hosipital
Department Center for Applied Bioinformatics
Street address 262 Danny Thomas Pl
City Memphis
State/province Tennessee
ZIP/Postal code 38105
Country USA
 
Platform ID GPL24676
Series (2)
GSE108115 UTX and 53BP1 co-regulate genetic programs for neural differentiation of human embryonic stem cells [RNA-seq]
GSE108116 UTX and 53BP1 co-regulate genetic programs for neural differentiation of human embryonic stem cells
Relations
BioSample SAMN10103244
SRA SRX4727779

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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