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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 06, 2019 |
| Title |
97PROL |
| Sample type |
SRA |
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| Source name |
urothelial organoid
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J.OlaHsd
tissue: bladder
cell type: urothelial organoid
sampleID: 97
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| Treatment protocol |
For differentiation experiments, NMU-o were cultured for the first 7 days in complete medium, reseeded (without disaggregation) in fresh Matrigel, and cultured either with complete medium or with differentiation medium (lacking WNTA3A and RSPO-1 conditioned medium, EGF, LY-2157299 and Noggin) for the following 7 days.
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| Growth protocol |
Normal mouse urothelial organoids (NMU-o) were maintained in culture with complete medium (proliferation condition), containing: Advanced DMEM/F12, 1x penicillin/streptomycin, 1x HEPES, 1x GlutaMAX, 50% WNT3A conditioned medium, 5% human RSPO1 conditioned medium, 1x N2 (Gibco, Cat. No. 17502048), 1x B27 (Gibco, Cat. No. 12587010), 50ng/mL human recombinant EGF (Invitrogen, Cat. No. PHG0311L), 1mM N-acetylcysteine (Sigma-Aldrich, Cat. No. 616-91-1), 50μg/mL human Noggin (Peprotech, Cat. No. 120-10C) and 1μM LY-2157299 (AxonChem, Cat. No. 700874-72-2). The ROCK inhibitor Y-27632 (Sigma-Aldrich, Cat. No. 129830-38-2) (10μM) was added during the first 3 days of culture.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated from Matrigel drops containing organoid-cultured cells using TRI Reagent® (Sigma-Aldrich, Cat. No. T9424) followed by the PureLinkTM RNA Mini Kit (Life Technologies, Cat. No. 12183020), according to manufacturer’s instructions. For 2D culture, RNA was extracted using ReliaPrepTM RNA Cell Miniprep System Kit (Promega, Cat. No. TM370). Samples were treated with DNase before reverse transcription (Life Technologies, Cat. No. AM1906).
PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in “TruSeq Stranded mRNA Sample Preparation Guide” ( Part #15031047 Rev. E). Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on a NovaSeq 6000- by following manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Image analysis and per-cycle basecalling was performed with Illumina Real Time Analysis software (RTA1.13). Conversion to FASTQ read format and sequence alignment with the ELAND algorithm (v2e) was performed with CASAVA-1.8 (Illumina).
Nextpresso 1.9 was used for RNAseq analysis (http://bioinfo.cnio.es/nextpresso/) (DOI: 10.2174/157489361266617081015385)
Nextpresso use tophat for alignments with these parameters useCuffmergeAssembly="false" nThreads="1" outputFormat="simple-table" libraryNormalizationMethod="geometric" Seed="123L" normalization="compatibleHits" reportSecondaryAlignments="false" bowtie="1" ReadEditDist="2" readGapLength="2" referenceIndexing="false"
Nextpresso use cufflinks for measure abundance with these parameters useGTF="true" nThreads="1" fragBiasCorrect="true" multiReadCorrect="false" libraryNormalizationMethod="classic-fpkm" maxBundleFrags="500000" normalization="compatibleHits" noEffectiveLengthCorrection="true" noLengthCorrection="false"
Nextpresso use cuffquant for measure abundance with these parameters useCuffmergeAssembly="false" nThreads="1" fragBiasCorrect="true" multiReadCorrect="false" seed="123L" maxBundleFrags="500000" noEffectiveLengthCorrection="true" noLengthCorrection="false" normalization="compatibleHits" noEffectiveLengthCorrection="true" noLengthCorrection="false"
Nextpresso use cuffnorm for measure abundance normalized for library size with these parameters useCuffmergeAssembly="false" nThreads="1" fragBiasCorrect="true" multiReadCorrect="false" seed="123L" maxBundleFrags="500000" noEffectiveLengthCorrection="true" noLengthCorrection="false" normalization="compatibleHits" noEffectiveLengthCorrection="true" noLengthCorrection="false"
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample from cuffnorm ; bigwig were generated with nextpresso 1.9
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| Submission date |
Jan 24, 2018 |
| Last update date |
Aug 06, 2019 |
| Contact name |
Enrique Carrillo de Santa Pau |
| E-mail(s) |
enrique.carrillo@imdea.org
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| Organization name |
Spanish National Cancer Research Centre (CNIO)
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| Department |
Cancer Cell Biology Programme
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| Lab |
Epithelial Carcinogenesis group
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| Street address |
C/ Melchor Fernández Almagro, 3
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| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE109566 |
Transcriptome analysis of differentiated normal mouse urothelial organoids reveals novel pathways activated during urothelial differentiation |
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| Relations |
| BioSample |
SAMN08390843 |
| SRA |
SRX3595400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2946666_Stem_97.bw |
60.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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