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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 21, 2022 |
| Title |
Mouse 2 somites |
| Sample type |
SRA |
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| Source name |
E11.5 somites and neural tube
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| Organism |
Mus musculus |
| Characteristics |
tissue: E11.5 somites and neural tube
strain: C57BL/6
treatment: untreated
seq method: nAnT-iCAGE sequencing (Murata 2014)
molecule: cDNA generated from 5'-cap selected mRNA (random hexamer RT)
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| Treatment protocol |
No treatment
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| Growth protocol |
C56BL/6J mice were used for timed mating; the date when a vaginal pug is present is assigned as embryonic day (E) 0.5 per convention.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were placed in dissection media (DMEM/F-12 1:1, 10% FBS, and 1% penn-strep; GIBCO) and chilled on ice in a 60 mm petri dish (Falcon) until dissection. Each embryo was sequentially transferred to a small dissection dish with Sylgard bottom (Living Systems Instrumentation) in the same media, pinned facing down by small dissection pins (Living Systems Instrumentation), and the neural tube and somites were dissected out using two angled scissors (2.5 mm and 4 mm cutting edge; FST) from the first somite to the tail end. Prior to transferring the dissected somite/neural tube to a 1.5 ml RNase-free microfuge tube (ThermoFisher Scientific), any remaining ventral endoderm-derived tissue was removed. To the dissected somite/neural tube (~100 µl, after removing as much media as possible), 400 µl of Trizol reagent (Invitrogen) was added. The tube was vortexed until the tissue was dissolved, and then immediately frozen on dry ice and stored at -80 °C until RNA isolation. Total RNA was isolated using the Direct-zol RNA Miniprep kit (Zymogen), followed by quantification and analyses using Nanodrop One (ThermoFisher Scientific) and Bioanalyzer (Agilent), respectively, and stored at -80 °C until use.
No-amplification non-tagging cap analysis of gene expression (nAnT-iCAGE) libraries were prepared using the cap-trap method as described in a detailed protocol (Murata et al., Methods Mol. Biol., 2014). In brief, 5 µg total mouse somite RNA was reverse transcribed (Superscript III, ThermoFisher) using random hexamer primers. Following cleanup with Agencourt RNAClean XP (Beckman Coulter), the RNA ends of RNA:cDNA hybrids were oxidized with NaIO4 (11.3 mM) on ice in the dark for 45 min, cleaned with Agencourt RNAClean XP, and biotinylated with biotin hydrazide (0.83 mM) at RT overnight. Single-stranded RNA was cleaved using RNAseONE ribonuclease and biotinylated RNA:cDNA hybrids were purified by incubation with Dynabeads M-270 streptavidin for 30 min, followed by washing and elution on a magnetic stand as previously described (Murata et al., Methods Mol. Biol., 2014)). Hybrid RNA was degraded with RNAseH, and any remaining free RNA was degraded with RNAse ONE. cDNA was concentrated in a centrifugal concentrator to ~5-10 µl and ligated to the pre-annealed double-stranded 5’linker containing a 6-nt barcode using ligation Mighty Mix at 16 °C for 16 hours. Following cDNA purification with Agencourt AMPure XP, cDNA was ligated to the pre-annealed 3’linker using ligation Mighty Mix at 16 °C for 16 hours. cDNA was treated with shrimp alkaline phosphatase and USER enzyme, followed by cleanup, second-strand synthesis using DeepVent DNA polymerase, primer degradation with Exonuclease I, and a final AMPure XP cleanup. Total cDNA was analyzed for quality and quantified using a Bioanalyzer (Agilent). DNA was pooled to a final concentration of 2 nM and sequenced on 2 lanes of a HiSeq FlowCell (Illumina) with 50 bp read length.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Barcode = CCTGAG at the 5' end of read
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| Data processing |
Library strategy: nAnTi-CAGE
Illumina HiSeq 2500
CAGE-seq reads were aligned to the mouse genome (mm10) using STAR v2.7.3a
vM24
Genome_build: Mus musculus mm10 (GRCm38)
Supplementary_files_format_and_content: WIGGLE (.wig) files contain chromosome mapping locations for the first non-barcode nucleotide of each read (mapped using STAR v2.7.3a). Signal values for each nucleotide position are normalized to reads per million (RPM)
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| Submission date |
Sep 21, 2021 |
| Last update date |
Feb 22, 2022 |
| Contact name |
James Saba |
| E-mail(s) |
jsaba1@jhmi.edu
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| Phone |
7038968025
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| Organization name |
Johns Hopkins
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| Department |
Molecular Biology and Genetics
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| Lab |
Rachel Green
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| Street address |
725 N Wolfe St, 714 PCTB
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE184515 |
Evolutionarily Conserved Inhibitory uORFs Sensitize Hox mRNA Translation to Start Codon Selection Stringency |
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| Relations |
| BioSample |
SAMN21536640 |
| SRA |
SRX12280971 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5591812_mouse2_CCTGAG_outputSignal.Unique.str1.out.wig.gz |
3.7 Mb |
(ftp)(http) |
WIG |
| GSM5591812_mouse2_CCTGAG_outputSignal.Unique.str2.out.wig.gz |
3.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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