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Sample GSM3208872

Query DataSets for GSM3208872

Status

Public on Jul 05, 2018

Title

Oncopeltus_fasciatus_buffer_RNA-seq_Head_individual34

Sample type

SRA

Source name

Head

Organism

Oncopeltus fasciatus

Characteristics

tissue: Head
developmental stage: Adult female post-mating
genotype: Carolina Biologicals

Treatment protocol

RNAi. Template for the in vitro transcription of reactions was prepared from a PCR reaction in which T7 phage promoter sequences were added to the gene-specific DNMT1 primers. For our control sequence, we used the red fluorescent protein (RED) sequence used in previous parental RNAi experiments in O. fasciatus. Sense and anti-sense RNA was synthesized in a single reaction using the Ambion MEGAscript kit (ThermoFisher Sci, Waltham, MA). After purification, the double-stranded RNA (dsRNA) concentration was adjusted to 2 μg/μL in injection buffer (5 mM KCl, 0.1 mM NaH2PO4). Females were injected with 5 μL of dsRNA between the abdominal sternites using an insulin syringe. Following injection, females were paired with an un-injected male to stimulate oviposition and fertilize eggs. Individual females with their mate were housed in petri dishes with sunflower seeds, water and cotton wool as an oviposition site. The parental RNAi protocol has been reported to result in 100% penetrance by the third clutch of eggs and this was also our experience. Eggs were collected between days 4–10 post-injection and assessed for development. Oncopeltus fasciatus embryos change from a creamy white color to orange as they develop, which indicates viability. Thus, color change is a useful tool for assessing healthy development. We examined the number of developing eggs at 5 days post-oviposition, at which point viable eggs are clearly distinguishable from inviable eggs, as well as hatching rate of eggs from females injected with double-stranded DNMT1 (dsDNMT1) and double-stranded RED (dsRED).

Growth protocol

Oncopeltus fasciatus cultures were originally purchased from Carolina Biologicals (Burlington, NC). Mass colonies were maintained in incubators under a 12 h:12 h light/dark cycle at 27°C. Colonies and individual experimental animals were fed organic raw sunflower seeds and provided with ad libitum deionized water. Late instar nymphs were separated from the mass colonies and housed under the same conditions. Nymph colonies were checked daily for newly emerged adults. Adults were separated by sex and kept with food and water for 7–10 days until females reached sexual maturity.

Extracted molecule

total RNA

Extraction protocol

Gene expression. RNA-seq libraries for RNA extracted from three DNMT1 knocked down and three control individuals were constructed using Illumina TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) following the manufacturer’s instructions with limited modifications. The starting quantity of total RNA was adjusted to 1.3 µg, and all volumes were reduced to a third of the described quantity.
Tissue was flash frozen in liquid nitrogen and stored at -80°C until processing. Total RNA and genomic DNA was extracted using a Qiagen RNAeasy micro kit (Qiagen, Venlo, The Netherlands) per manufacturer’s instructions with Qiagen Qiazol as the lysis buffer. Complementary DNA (cDNA) was synthesized from 500 ng RNA with qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

11 days post-injection of buffer (control)
TS_34_Head_buffer_RNA_S24_R1_001
processed data file:
head.RNAprocessed.ds-dnmt1_16_24_26.control_1_2_34.xlsx

Data processing

Gene expression. Raw RNA-seq FASTQ reads were trimmed for adapters and preprocessed to remove low-quality reads using Trimmomatic v0.33 (arguments: LEADING:10 TRAILING:10 MINLEN:30) prior to mapping to the O. fasciatus v1.1 genome assembly. Reads were mapped using TopHat v2.1.1 supplied with a reference General Features File (GFF) to the O. fasciatus v1.1 genome assembly, and with the following arguments: -I 20000 --library-type fr-firststrand --b2-very-sensitive.
genome_build: O. fasciatus: v1.1 (https://www.hgsc.bcm.edu/arthropods/i5k), B. germanica: v1 (https://www.hgsc.bcm.edu/arthropods/i5k)
Supplementary_files_format_and_content: Tab delimited; please see metadata file for column headers.

Submission date

Jun 20, 2018

Last update date

Jul 05, 2018

Contact name

Robert J Schmitz

E-mail(s)

[email protected]

Organization name

University of Georgia

Department

Genetics