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| Status |
Public on Apr 03, 2020 |
| Title |
C11d0_2 |
| Sample type |
SRA |
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| Source name |
C11-CRL2429 iPS day 0
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| Organism |
Homo sapiens |
| Characteristics |
cell type: undifferentiated human iPS cells
atcc cell line source: CCD-1112Sk (ATCC CRL-2429)
gender: male
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| Treatment protocol |
N/A
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| Growth protocol |
hESC and iPSC were grown in hESC culture medium (80% DMEM F-12, 20% KnockOut-Serum replacement, 2 mM 1-glutamine, 1% non-essential amino acids (NEAA) 0.1 mM 2-mercaptoethanol and between 50-100 ng/ml basic fibroblast growth factor) at 37°C at 5% CO2 at high humidity. Cells were maintained on Mouse embryonic fibroblast feeder layers supplied by the Australian Stem Cell Centre. For experimentation, cells were cultured in feeder-free conditions on Matrigel (BD) in MEF conditioned hESC media. Cells were passaged as previously described before replating at a seeding ratio of between 1:2 and 1:6. hESC media was replaced daily and cells were split at approximately 80% confluence on days 6-7. All work was carried out with informed consent from patients under the approval of the Human Research Ethics Committee (HREC/09/QRCH/103). Embryoid bodies (EBs) were formed by detachment of undifferentiated iPS cells from the MEF monolayer (12,000/cm2) using a P200 pipette. EBs were transferred to a low-attachment plate (Costar) in Knock-Out Serum (KSR) replacement hESC culture medium (80% DMEM -F12 (GIBCO), 20% KnockOut-Serum replacement (GIBCO), 2 mM L-glutamine (GIBCO), 1% non-essential amino acids (NEAA) (GIBCO), 0.1 mM 2-mercaptoethanol at 37°C at 5% CO2 at high humidity. EBs were cultured in the absence of basic fibroblast growth factor (to promote differentiation towards neurectodermal lineage). Medium was changed each day. After four days EBs were resuspended in neural induction medium (DMEM F-12with Glutamax (GIBCO), N2 supplement (GIBCO) and heparin sulfate (R&D)) and supplemented with human FGF8 (Sigma Aldrich) (100 ng/mL) and RA (Retinoic acid) (10 µM) (Sigma Aldrich). After 7 days of incubation in this medium EBs were deposited in plates coated with laminin (Sigma) (2 µg/mL) and fibronectin (BD) (5 µg/mL) in basal medium eagle (BME) media (Invitrogen) supplemented with ITS (Insulin, Transferrin, Selenite) (GIBCO), human FGF8 (100 ng/mL), human FGF4 (Life technologies) (100 ng/mL) and human basic fibroblast growth factor (Invitrogen) (20 ng/mL) and allowed to grow for four days. Medium was changed to DMEM-F12 with human FGF8 (100 ng/mL), WNT1 (Peprotech/Abacus) (50 ng/mL) and WNT3A (50 ng/mL) (R&D) for five days. The next stage of differentiation involved culturing the cells in BME media supplemented with N2, B27 (GIBCO), human BMP4 (R&D), (50 ng/mL), human BMP6 (R&D) (20 ng/mL), human BMP7 (Peprotech/Abacus) (100 ng/mL) and human growth differentiation factor 7 (GDF7) (R&D) (100 ng/mL) for a further eight days. Attached cells were then disaggregated and replated on laminin/fibronectin for another 8 days of growth in BME supplemented with N2, B27, human BMP4 (50 ng/mL), human BMP6 (20 ng/mL), human BMP7 (100 ng/mL), human GDF7 (100 ng/mL), human Sonic hedgehog (R&D) (100 ng/mL), human neurotrophin 3 (R&D), (NT3) (100 ng/mL), jagged1 (JAG1) (R&D) (20 ng/mL) and human brain derived neutrotrophic factor (BDNF) (100 ng/mL) (R&D).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNA Spin II isolation columns (Machery Nagel). On column digestion of DNA with RNase free DNase was performed according to the manufacturer’s specifications (Ambion, Austin, TX). RNA samples were subject to RNA integrity analysis using the RNA 6000 Nano total RNA kit (Agilent). All samples recorded a RIN (RNA integrity number) in excess of 8.5 out of 10. Samples were processed according to the TruSeq Stranded Total RNA Sample preparation LS protocol kit (Illumina) as per the manufacturer’s specifications.
Total RNA libraries were prepared for sequencing using standard Illumina protocols in the TruSeq Total Stranded Library Preparation kit. 100 bp PE reads were generated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
day0. This is the same iPS line as SAMD00005689, but the passage, neuronal differentiation, and RNA purification are all distinct.
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| Data processing |
Illumina Casava software used for basecalling.
After read quality control using FastQC (Andrews, 2012), primary alignment to the reference human genome (hg19) was carried out using the Burrows-Wheeler Aligner (Li and Durbin, 2009) version 0.6.2 with default mapping parameters.
Output data was converted to bam format using SAMtools (Li et al., 2009).
The exon_utils.py module of the MISO framework (Katz et al., 2010) was used to extract constitutive exons longer than 1000 nucleotides, and pe_utils.py was used to determine the mate inner distance and mate standard deviation parameters for each dataset (these are provided in the table below).
Tophat (Kim et al., 2013) version 2.0.8 was then used to map reads to the genome and reference transcriptome (GENCODE v. 17 (Harrow et al., 2012)) simultaneously, with the parameters --b2-very-sensitive --read-mismatches 3 --read-gap-length 2 --read-edit-dist 3 --read-realign-edit-dist 0.
All subsequent analyses were conducted using the results of this mapping. Count tables were obtained using the HTSeq (Anders, 2010) framework in union mode, with GENCODE v. 17 as a reference.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file reports gene-level count table for each GENCODE17 annotated gene
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| Submission date |
Jan 29, 2015 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Darya Pavlovna Vanichkina |
| E-mail(s) |
d.vanichkina@gmail.com
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| Phone |
+61 (0)4 20889939
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| Organization name |
University of Sydney
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| Street address |
32 Queen Street
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| City |
Chippendale |
| State/province |
New South Wales |
| ZIP/Postal code |
2008 |
| Country |
Australia |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE65423 |
Transcriptomic characterization of a mixed population of pluripotent stem cell derived cerebellar neuron-like progenitors |
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| Relations |
| BioSample |
SAMN03316890 |
| SRA |
SRX858049 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1595893_C11d0_2.counttable1.txt.gz |
237.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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