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Sample GSM2584534

Query DataSets for GSM2584534

Status

Public on Jul 11, 2018

Title

Lilium_5mm_mRNA-seq

Sample type

SRA

Source name

Lilium maculatum - Premeiotic anther

Organism

Lilium maculatum

Characteristics

tissue: anther

Growth protocol

Flowering Lilium plants were purchased from Home Depot (Newark, Delaware)

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Lilium maculatum L-5, 5 mm anthers

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). SMRT sequencing libraries were processed using SMRT Analysis software (version 2.3) from Pacific Biosciences with recommended settings.
Genome_build: Data from mRNA-seq and SMRT-seq were used for de novo transcriptome assemblies, so as to compensate for the lack of Lilium genome. These assemblies were then used along with sRNA data to identify phased-siRNA precursor transcripts and other protein factors that play a role in reproductive phasiRNA pathways. mRNA-seq assembly was performed using “Trinity RNA-Seq assembler”, full-length transcript assembly was prepared using “SMRT-tools” (v3) provided by Pacific Biosciences. Phased siRNA precursors were identified using “PHASIS suite” (https://github.com/atulkakrana/PHASIS/wiki), and transcripts for protein-coding genes were identified using “Trinotate” (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.

Submission date

Apr 19, 2017

Last update date

May 15, 2019

Contact name

Blake C. Meyers

E-mail(s)

[email protected]

Phone

314-587-1422

Organization name

Donald Danforth Plant Science Center