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Sample GSM3747600 Query DataSets for GSM3747600
Status Public on Oct 04, 2019
Title D8 A
Sample type SRA
 
Source name Boundary organoid
Organism Homo sapiens
Characteristics cell type: iPSC derived boundary organoid
region: Anterior part of micro-dissected organoid
culture period: day8 from undifferentiated iPSC
Growth protocol colonies of hiPSCs were isolated in Accutase and 150,000 cells/mL were plated on Matrigel coated tissue culture plate. Medium was changed to RPMI 1640 medium containing 100 ng/mL Activin A and 50 ng/mL bone morphogenetic protein 4 at Day 1, 100 ng/mL Activin A and 0.2 % fetal calf serum at Day 2 and 100 ng/mL Activin A and 2% FCS at Day 3. For Day 4-7, cells were cultured in gut growth medium (Advanced DMEM/F12 with 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin, B27 and N2) supplemented with 200 ng/mL noggin, 500 ng/ml fibroblast growth factor 4 and 2 µM CHIR99021 for anterior gut cell induction and supplemented with 500 ng/ml FGF 4 and 3 µM CHIR99021 for posterior gut cell induction. Cultures for cell differentiation were maintained at 37°C in an atmosphere of 5% CO2 / 95% air and the medium was replaced every day. On Day 7, anterior or posterior gut cells were dissociated to single cells by incubation with TrypLE Express at 37°C. Cells were centrifuged at 1000 rpm for 3 minutes and, after removing supernatant, the pellet was re-suspended in gut growth medium containing 10 uM of Y-27632 dihydrochloride. The anterior or posterior gut cell suspensions were plated on 96 well round bottom ultra-low attachment plate at density of 10,000 cells/well and incubated at 37°C for 24 hours to form spheroid. On Day 8, generated single anterior spheroid and posterior spheroid were mixed on 96 well round bottom ultra-low attachment plate in gut growth medium for 24 hours to form fused boundary spheroids (A-P spheroids). On Day9, A-P spheroids were embedded in Matrigel drop and were cultured in gut growth medium to generate multi-organ organoids.
Extracted molecule total RNA
Extraction protocol RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany).
Sample preparation for RNA sequencing was performed using SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories) according manufacture’s user manual.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing Illumina OLB 1.9.4 software used for basecalling and bcl conversion/demultiplexing/fastq creation.
Transcripts Per Million (TPM) were quantified using kallisto v0.44.0
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample.
 
Submission date May 03, 2019
Last update date Oct 04, 2019
Contact name Norikazu Saiki
E-mail(s) nrsk.ior@tmd.ac.jp
Phone +81-466-32-1904
Organization name Tokyo Medical and Dental University
Department Institute of Research
Street address 1-5-45 Yushima, Bunkyo-ku
City Tokyo
ZIP/Postal code 113-8510
Country Japan
 
Platform ID GPL16791
Series (1)
GSE121830 RNA-sequencing with micro-dissected boundary organoid into anterior, posterior, and boundary regions
Relations
BioSample SAMN11569573
SRA SRX5785474

Supplementary file Size Download File type/resource
GSM3747600_D8A_human37_Gene_TPM.xls.gz 257.5 Kb (ftp)(http) XLS
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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