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| Status |
Public on Jul 15, 2024 |
| Title |
gMCE4 |
| Sample type |
SRA |
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| Source name |
Brassica napus endosperm
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| Organism |
Brassica napus |
| Characteristics |
cultivar: Topas
tissue: Brassica napus endosperm
genotype: Brassica napus cv. Topas
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| Treatment protocol |
Flowers were hand pollinated at the time of anthesis during the times of 14:00 - 17:00. Siliques were collected at 0 (ovule), 7 (globular), 10 (heart), and 28 (mature green) days after pollination. Tissue was fixed in 1:3 (v/v) glacial acetic acid: 85% ethanol for 24 h at 4°C. Tissue was dehydrated in a graded ethanol series, then infiltrated with molten parrafin wax at 60°C. Tissue was then embedded in 100% paraffin wax and 7 µm sections were cut onto polyethylene napthalate membrane slides for LMD. Sections were deparaffinized and subregions were dissected and isolated out using LMD.
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| Growth protocol |
B. napus plants were grown in growth chambers nder long day cycles (16 h light, 8 h dark, 22°C, RH 50%–70%)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Once dissection was completed, total RNA was extracted using Ambion RNaqueous micro kit with Qiagen RNAse-free Dnase to remove genomic DNA. RNA quality was evaluated using an Agilent Bioanalyzer. Minimum RIN of 3 was required for further processing.
Fragmentation was conducted via Covaris ultra-sonicator. Library preparation was done with NuGen Ovation Multiplex System. Libraries were quantified and assessed in quality via Quant-iT PicoGreen dsDNA assay kit, qPCR, and the High Sensitivity DNA analysis kit (Agilent) on the Agilent 2100 Bioanalyzer. Libraries were pooled and size selected at 350-600 bp on an E-Gel (Invitrogen). Samples were sequenced on the Illumina HiSeq 2000 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
low quality reads were trimmed and aligned to the B. napus cv. Topas reference genome using hisat2. Quantification of uniquely mapped reads were done using the featurecounts package. Counts were normalized to TPM.
Assembly: Brassica napus cv. Topas
Supplementary files format and content: Tab delimited text file of normalized TPM values. Raw data uploaded to SRA.
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| Submission date |
Jul 09, 2024 |
| Last update date |
Jul 15, 2024 |
| Contact name |
Mark Belmonte |
| Organization name |
University of Manitoba
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| Department |
Biological Sciences
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| Street address |
50 Sifton Rd.
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| City |
Winnipeg |
| State/province |
Manitoba |
| ZIP/Postal code |
R3T 2N2 |
| Country |
Canada |
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| Platform ID |
GPL15579 |
| Series (1) |
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| Relations |
| BioSample |
SAMN39463218 |
| SRA |
SRX23253734 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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