RNA-seq (Drosophilidae)

Knowledge graph centered on RNA-seq (Drosophilidae) with 68 nodes and 161 connections. Top connected: Beetles, Hymenoptera, Lepidoptera, Diptera, Boechera stricta.

Method synopsis

Illumina GAIIx RNA sequencing of larval samples from different plant treatments followed by differential expression analysis to identify genes responsive to plant chemical defenses.

Synthesized from method descriptions across 2 papers using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Genes involved in the evolution of herbivory by a leaf-mining drosophilid fly (2012), Genome Biology and Evolution. The protocol was originally introduced by Mining the plant-insect interface with a leafmining <i>Drosophila</i> of <i>Arabidopsis</i> (2011), Molecular Ecology. Implementations in other papers (listed below) may differ.

1. RNA extraction and library preparation
   Four larval pools were obtained: two pools from Col-0 plants (pool 1: N=12, pool 2: N=18 larvae) and two from GKO plants (pool 3: N=12, pool 4: N=12 larvae). Larvae were flash frozen in liquid nitrogen and crushed in Qiagen Tissue Lysis Buffer. RNA extraction followed with 500 μl REAGENT per sample using RNA Nano Chip to determine RNA integrity.
   Quantities: 4 RNA pools total: 12, 18, 12, 12 larvae per pool respectively, 500 μl REAGENT per sampleDuration: Not specifiedConditions: Samples flash frozen in liquid nitrogen
   Equipment: Qiagen Tissue Lysis Buffer, gDNA Eliminator spin column, Agilent 2100 Bioanalyzer, RNA Nano Chip
2. Illumina transcriptome sequencing
   Illumina mRNA sequencing sample prep kit was used to prepare libraries for each of four RNA pools, resulting in four RNA-seq libraries subjected to clustering and 72 bp sequencing on an Illumina GAIIx instrument. Libraries were not normalized.
   Quantities: 4 RNA-seq libraries, 72 bp read lengthDuration: Not specifiedConditions: Not specified
   Equipment: Illumina mRNA sequencing sample prep kit, Illumina GAIIx sequencer
3. Transcriptome assembly and analysis

Typical Equipment

• Qiagen Tissue Lysis Buffer
• gDNA Eliminator spin column
• Agilent 2100 Bioanalyzer
• RNA Nano Chip
• Illumina mRNA sequencing sample prep kit
• Illumina GAIIx sequencer
• AbySS version 1.2.1 software

Output Measurements

• gene expression levels
• differentially expressed genes

Papers Using This Protocol (2)

Species Studied With This Protocol

Places Where Used