GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3072219

Query DataSets for GSM3072219

Status

Public on Apr 12, 2018

Title

Nicotiana bonariensis Stage 6 replicate 3

Sample type

SRA

Source name

Nectary tissue

Organism

Nicotiana bonariensis

Characteristics

tissue: nectary
developmental stage: stage 6

Extracted molecule

total RNA

Extraction protocol

Six species of tobacco (Nicotiana rustica [PI-34752; Accession TR-16]; Nicotiana glauca [PI-282690; Accession TW-54]; Nicotiana benthamiana [PI-555684; Accession TW-17]; Nicotiana clevelandii [PI-555490; Accession TW-31]; Nicotiana plumbaginifolia [PI-302476; Accession TW-107]; Nicotiana bonariensis [PI-555489; Accession TW-28]) seeds were obtained from Tobacco Germplasm Collection North Carolina State University Oxford NC 27565 (https://npgsweb.ars-grin.gov/). These seeds were initially sewn in a single 24-cm pot of Sungro professional growing mix at high density. After approximately 20 days, the seedlings reached ~15 cm tall. Subsequently individual plants were transplanted to separate 30-cm pots of Sungro mix. The plants were grown to maturity under 16 h day/ 8 h night conditions until flowering. Flowers were staged as described in Koltunow, et al., [Plant Cell 2(12): 1201-1224(1990)]. RNA samples were isolated from floral nectaries at floral stages: Stage 6 [immature; beginning of metabolic switch]; Stage 9 [immature; pre-secretory], Stage 12 [mature flowers at anthesis] and Stage PF [post-fertilization]. All nectary tissues were manually dissected by hand with the RNA being immediately extracted by mechanical disruption with a microcentrifuge pestle and using an RNAqueous® RNA isolation kit (Ambion, Austin, TX) with Plant RNA Isolation Aid (Ambion, Austin, TX). Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality for all samples prior to submission to the University of Minnesota Genomics Center for mRNA isolation, barcoded library creation and Illumina HiSeq 2500 sequencing. Seventy-five TruSeq RNA v2 libraries were created (triplicate samples for nectaries at four timepoints each from six independent species, plus 3 control libraries from Nicotiana plumbaginifolia (leaves, ovaries, and stems) and sequenced via 50 bp, paired-end runs on the HiSeq 2500 using Rapid chemistry. All libraries were pooled and sequenced across two full lanes. This generated 200 M reads for each lane and the average quality scores were above Q30.
Illumina’s Truseq RNA Preparation v2 kit (RS-122-2001)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Contigs assembled with Trinity v.2.3
Reads aligned to contigs using NCBI blastn
Contigs mapped to Nicotiana tabacum mRNA, and secondarily to Arabidopsis thaliana. Read counts for mapped contigs were tested for differential expression between stages using DESeq in R.
Supplementary_files_format_and_content: Nicotiana_Means_SDs_DEb.xls: Mean contig read counts, differential expression test results, and associations by similarity to Arabidopsis thaliana genes

Submission date

Mar 30, 2018

Last update date

Apr 12, 2018

Contact name

Marshall Hampton

Organization name

University of Minnesota Duluth

Street address

1117 University Drive

City

Duluth