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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 27, 2023 |
| Title |
smallRNA_Htt WT mESC replicate 1 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
genotype: wild type
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| Treatment protocol |
Self-renewing mES cells were dissociated and plated onto 0.1% gelatin-coated tissue culture plastic at a density of 0.5–1.5 104/cm2 in N2B27 medium. N2B27 is a 1:1 mixture of DMEM/F-12 (Thermo Fisher Scientific) supplemented with N-2 supplement (Gibco) and Neurobasal medium (Thermo Fisher Scientific) complemented with B27 supplement (Thermo Fisher Scientific).The medium was renewed every other day. After 7 days, cells were detached using Accutase (Thermo Fisher Scientific) and plated on 3 µg/ml laminin-coated tissue culture dishes in NS expansion medium. Mouse neural progenitor cells were routinely passaged 1:2-1:4 every 3-5 days using Accutase and maintained in NS medium, composed by Euromed-N (Euroclone) supplemented with 1:1000 FGF (R&D) and EGF (Sigma), 1% N-2 Supplement (Gibco), 2 mM L-glutamine (Gibco) and 100 U/ml Penicillin/Streptomycin (Gibco) on laminin-coated plates (Sigma, 3.5 µg/ml).
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| Growth protocol |
The allelic series of heterozygous Hdh CAG knock-in mouse embryonic stem cell (mESC) lines, consisting of 4 alleles of different Htt CAG size (20, 50, 89 and 111 CAG) were kindly given by Dr. Marcy E. MacDonald (Massachussets General Hospital and Harvard Medical School, Boston, US). They were cultured in KnockOut DMEM (Gibco), supplemented with 15% of ES cell FBS (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml Penicillin/Streptomycin (Gibco), 1% Non-essential Amino Acids (Gibco), 0.1 mm 2-mercaptoethanol (Sigma) and 1000 U/ml of leukemia inhibitory factor (LIF) (Voden), on plates coated with 0.1% gelatin (Millipore) and with a feeder layer of CF-1 IRR MEF (TebuBio). Differentiation of mESC to mNPC was performed as described in Conti et al., PLoS Biology 2005. Both mESCs and mNPCs were incubated at 37°C and 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cell lines by using TRIzol reagent (Invitrogen) following manufacturer's instructions.
The RNA samples were subjected to DNAse I treatment (Ambion) according to manufacturer instructions . RNA library preparation was obtained using the standard Illumina protocols, and sequenced on a Illumina HiSeq 2500 machine using single-end, 50bp reads, obtaining approx 15M reads/sample.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
HttWTESCa
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| Data processing |
Illumina Casava 1.8 software was used for basecalling and demultiplexing.
Sequenced reads were trimmed for adaptor sequence and low-quality nucleotides, then aligned against the mouse Gencode v23 transcriptome using STAR (genes) with default parameters and quantified with htseq-count
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: tab-delimited text files include raw read counts for each Sample
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| Submission date |
May 27, 2021 |
| Last update date |
Sep 27, 2023 |
| Contact name |
Erik Dassi |
| E-mail(s) |
erik.dassi@unitn.it
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| Organization name |
University of Trento
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| Department |
CIBIO
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| Street address |
Via Sommarive, 9
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| City |
Trento |
| State/province |
TN |
| ZIP/Postal code |
38123 |
| Country |
Italy |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE175657 |
CAG repeat expansion in the Huntington’s Disease gene correlates with defective linear and back-splicing [smallRNA-seq] |
| GSE175658 |
CAG repeat expansion in the Huntington’s Disease gene correlates with defective linear and back-splicing |
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| Relations |
| BioSample |
SAMN19368330 |
| SRA |
SRX11005888 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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