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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 09, 2021 |
| Title |
tKO26b/a1/a2 d15 neural cell miR-26a1/a2/b knockout CCC55 |
| Sample type |
SRA |
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| Source name |
Neural cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived neural cell
genotype: miR-26a1/a2/b knockout CCC55
strain: R1
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| Growth protocol |
ESCs were differentiated into the neural lineage as described (Bibel et al., 2007b; Wolber et al., 2013) with slight modifications. Briefly, 3x106 ESCs were plated into 10 cm petri dishes (Greiner Bio One) and cultured for 5 days under floating conditions in ESC medium with 10% FCS and without LIF to allow for the formation of embryoid bodies (EBs). To generate attached EBs (att. EBs), EBs in floating conditions were transferred to tissue culture dishes on day 5 and cultured in serum-free neural selection ITS-medium (DMEM/Ham’s F-12, 100 U/ml penicillin/streptomycin, 2mM L-glutamate, 5 μg/ml insulin, 50 μg/ml transferrin, 30 nM sodium selenite, 2.5 μg/ml fibronectin, and 1 μM retinoic acid). After 4 days, cells were trypsinized and further cultured (1,5x105 cells/cm2) on polyornithine- and laminin-coated dishes or coverslips in N2 medium (DMEM/Ham’s F-12, 100 U/ml penicillin/streptomycin, 2 mM L-glutamate, 25 μg/ml insulin, 50 μg/ml transferrin, 30 nM sodium selenite, 20 nM progesteron, and 100 μM putrescin). Medium was replaced 2h and 24h post plating. Thereafter NPCs were cultured in Neurobasal medium (Thermo-Fisher), supplemented with 100 U/ml penicillin/streptomycin, 2mM L-glutamate, and 2% B27 supplement (Thermo-Fisher). Medium was changed every 2nd day. Cells were cultured up to 15 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from two independent clones of WT and tKO26b/a1/a2 cell lines and two biological replicates of NCs (day 15) were isolated using peqGold RNA pure (PeqLab). rRNA was removed using the Ribo-Zero rRNA Removal Kit (Illumina).
cDNA library preparation using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). For small RNA sequencing total RNA was isolated and a 3´barcoded adapter was ligated usig truncated T4 RNA ligase (WT_R1_A: 5´-GTAGAGTGACTGTGGAATTCTC-3`; WT_R1B:5´-GTAGAGTCTGTGTGGAATTCTC-3´; WT_R1C:5´-GTCCGCTGACTGTGGAATTCTC-3´; WT_R1D: 5´- GTCCGCTCTGTGTGGAATTCTC-3´; CCC_22A: 5´-GTAGAGGCGATATGGAATTCTC-3´; CCC_22B: 5´-GTCCGCTCATAGTGGAATTCTC-3´; CCC_55A: 5´-GTAGAGTCATAGTGGAATTCTC-3´; CCC_55B:5´-GTCCGCGCGATATGGAATTCTC-3´). Ligated RNAs were pooled and size selected. Small RNA cDNA libraries were generated using SuperscriptIII (Invitrogen) and sequenced by Illumina Sequencing. (Hafner et al., 2010)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequence reads were aligned to the mouse genome (version mm10) using TopHat (Trapnell et al., 2012).
Transcripts were quantified by Cufflinks and Cuffdiff and differential expression was determined (Trapnell et al., 2012).
Small RNA seq reads were mapped against murine miR annotation database (miRBase) using Burrow-Wheeler aligner. Each miR profile was normalized to relative read frequencies (Farazi et al., 2012).
Genome_build: mouse genome (version mm10)
Supplementary_files_format_and_content: tab-delimited text files include miR Sequencing reads or output of cuffdiffs
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| Submission date |
Apr 08, 2021 |
| Last update date |
Apr 09, 2021 |
| Contact name |
Nina Was |
| E-mail(s) |
nina.houben@uni-wuerzburg.de
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| Organization name |
Universität Würzburg
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| Department |
Institut für medizinische Strahlenkunde und Zellforschung
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| Lab |
AG Becker
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| Street address |
Zinklesweg 10
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| City |
Würzburg |
| State/province |
Bavaria |
| ZIP/Postal code |
97078 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE171727 |
The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST |
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| Relations |
| BioSample |
SAMN18675060 |
| SRA |
SRX10556660 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5231640_Cuff_diff_CCC55.diff.gz |
1.2 Mb |
(ftp)(http) |
DIFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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