title: Juvenile
organism: Heterodera avenae
description: Protocols: Cysts of H. avenae were collected from infested wheat roots growing in the plots of the Indian Agricultural Research Institute, New Delhi, India. Genetic homogeneity of the population was determined by PCR-RFLP and sequencing of ITS (Internal Transcribed Spacer) regions of rDNA (ribosomal DNA). A genetically homogenous population was then multiplied on wheat plants grown in pots kept in a greenhouse (PMID:23390348). Newly formed cysts were collected manually from the wheat roots using a dissecting microscope and kept for hatching. Hatched J2s were collected and later used for RNA extraction. Likewise, hatched J2s were also used to infect wheat roots and feeding females (FF) that developed and protruded from roots 6 to 8 weeks after infection were collected, frozen (-80 degrees C) for later RNA extraction. Total RNA was extracted from the frozen J2s and FFs using TRIzol (Invitrogen Life Technologies) according to the manufacturer's instructions. Extracted RNA was assessed for quality and quantity using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA with an RNA integrity number (RIN) of 8.0 was used for mRNA purification. mRNA was purified from 1 ug of intact total RNA using oligodT beads (TruSeq RNA Sample Preparation Kit, Illumina). The purified mRNA was fragmented at elevated temperature (90 degrees C) in the presence of divalent cations and reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) by priming with random hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RNaseH. The cDNA was cleaned using Agencourt Ampure XP SPRI beads (Beckman Coulter).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-01-27
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2014-01-24
attributes.tag: External Id
attributes.value: SAMEA2338038
attributes.tag: INSDC center alias
attributes.value: Indian Agricultural Research Institute
attributes.tag: INSDC center name
attributes.value: Indian Agricultural Research Institute
attributes.tag: INSDC first public
attributes.value: 2014-01-27T17:00:58Z
attributes.tag: INSDC last update
attributes.value: 2014-01-24T16:04:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2221:Juvenile
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: juvenile
attributes.tag: phenotype
attributes.value: pre-parasitic
attributes.tag: sample name
attributes.value: E-MTAB-2221:Juvenile
attributes.tag: sex
attributes.value: male