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| Status |
Public on Jul 07, 2018 |
| Title |
05_ant_bud_v-dsRNA |
| Sample type |
SRA |
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| Source name |
Asparagus dsRNA, <0.5 mm anthers (whole buds), BM14-181, Rnase V1
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| Organism |
Asparagus officinalis |
| Characteristics |
tissue: floral bud
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| Growth protocol |
Flowering Asparagus plants were harvested from T.S. Smith and Sons Farms (Bridgeville, Delaware)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Anthers were dissected using a 2 mm stage micrometer in a stereo microscope, and immediately frozen in liquid nitrogen until total RNA isolation was performed. Total RNA was isolated using the PureLink Plant RNA Reagent following the manufacturer’s instructions. Total RNA quality and quantity were assessed before proceeding to the next step.
Small RNAs (20 to 30 nt) were size selected in a 15% polyacrylamide/urea gel and libraries were constructed using the TruSeq Small RNA Prep Kit (Illumina, cat. # RS-200-0012). Small RNA libraries containing different index tags were pooled and sequenced on an Illumina HiSeq 2500 instrument at the Delaware Biotechnology Institute. The complete protocol for sRNA library preparation is described in Mathioni et al. (DOI: 10.1002/cppb.20043). RNA-seq libraries (for mRNA) were constructed using the TruSeq Stranded Total RNA Library Preparation Kit with Ribo-Zero Plant (Illumina, cat # RS-122-2401), and RNA was treated with DNase I (NEB, cat # M0303S) and then cleaned using the RNA Clean & Concentrator™-5 (Zymo Research, cat # R1015). Single Molecule Real Time (SMRT) sequencing libraries were prepared using protocol recommended by Pacific Biosciences for Isoform Sequencing using SageELF size selection system
Small RNA sequencing using Illumina single-end (1x50 bp) reads. Messenger RNA sequencing using Illumina single-end (2x150 bp) reads and Single Molecule Real Time (SMRT) sequencing of full length (cDNA) transcripts.
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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 |
Asparagus dsRNA, <0.5 mm anthers (whole buds), BM14-181, Rnase V1
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| Data processing |
Sequenced reads were trimmed to remove adapters and converted to flat file format using the pre-processing script described in Mathioni et al. (DOI: 10.1002/cppb.20043). Processed files for sRNA libraries were aligned to the Asparagus genome (v.1) using Bowtie (v0.12.8) with no allowed mismatches. Mapped sRNA reads were finally normalized to empirically derived, 30 s. Raw SMRT-sequencing data was pre-processed using the pbscript-tofu tool set (v2.3.0) from Pacific Biosciences using the default settings. The pre-processing included generation of subreads FASTA file using the RS_subreads protocol, followed by classification of reads to full-length and non-full-length categories, clustering of transcripts to consensus isoforms by ICE algorithm and final polishing by Quiver algorithm (min. accuracy = 0.99). For all downstream analysis, “high QV” transcript set generated from Quiver analyses was used. mRNA-seq assembly was performed using “Trinity RNA-Seq assembler”.For this, reads from paired-end libraries were first combined into two (FASTQ) files, one corresponding to left reads and other to right reads. Reads from the single-end libraries were then added to the combined left reads (FASTQ) file. These left and right reads files were used to generate a hybrid assembly with the default settings except for the minimum assembled contig length (set to 250 nt). ExN50 and the quality of assemblies was accessed as recommended in Trinity workflow. Transcripts from de novo assembly were annotated using Trinotate workflow with the default settings (https://trinotate.github.io/).
Supplementary_files_format_and_content: Flat file with tab separated reads and abundance counts for sRNAs, FASTA files for SMRT-seq and FASTA files for Illumina mRNA seq. Since the mRNAseq and SMRT-seq samples were used for transcriptome assembly, there are no quantitative data for these samples. The transcriptome assembly made from the mRNA-seq samples is available on the series record.
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| Submission date |
Apr 06, 2018 |
| Last update date |
Jul 08, 2018 |
| Contact name |
Blake C. Meyers |
| E-mail(s) |
bmeyers@danforthcenter.org
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| Phone |
314-587-1422
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| Organization name |
Donald Danforth Plant Science Center
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| Lab |
Meyers lab
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| Street address |
975 N Warson Road
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63132 |
| Country |
USA |
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| Platform ID |
GPL24857 |
| Series (1) |
| GSE112800 |
Small RNA (sRNA) sequencing data from pre-meiotic and meiotic anthers of Asparagus |
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| Relations |
| BioSample |
SAMN08875180 |
| SRA |
SRX3897637 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3083930_5885_chopped.txt.gz |
17.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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