Meloidogyne chitwoodi
Protocols: Isolation of eggs: Heavily infected root balls have been obtained from a M. chitwoodi greenhouse culture. Root balls were washed gently free of sand on a top 1000, 175, 105, 45 and 25 µm to bottom. The sieves were rinsed from top to bottom and the eggs collected on the 25 µm sieve, were rinsed with tap water to wash away any remaining Na-hypochloride. Eggs were further purified over 35% sucrose and washed 3x with tap water.  Hatching J2 of M. chitwoodi: Sucrose purified egg-suspension was treated as follows: Eggs were incubated in a 0.02% sodium azide in tap water solution, at 200 rpm at 20°C for 20 minutes. The egg suspension was poured onto a 25 µm sieve and the eggs were rinsedwell with tap water to remove all traces of sodium azide. Sieve with sodium azide treated eggs was placed in a 14 cm glass petri dish, ca. 30 mL of a solution containing 1.5 mg/ml gentamycin and 0.05 mg/ml nystatin in sterile tap water was poured onto the sieve in the petri dish. Petri dishes with eggs on 25 µm sieves were placed in a transparent shoebox in the dark at room temperature (ca. 20°C) After 5 days the hatched juveniles (J2) were collected from the petri dish, and sucrose purified and washed 3x with tap-water.  Both eggs and J2 sample were RNA-later treated for 30 minutes at room temperature before storage at -80°C.  Upon removal from freezer directly added 2 stee1000 µm sieve and cut in pieces. Root material was submerged in 0.5% Na-hypochloride solution and shaken in a plastic bottle for 3 minutes. Sodium hypo-chloride treated root material was pored over a stack of sieves: From l balls 3 mm and retched 1x 2 minutes and 1x 1 minute at 30/s in a Tissue lyser.  From this point onwards RNA extraction was performed according to the Maxwell LEV16 plant RNA kit (Promega) protocol as provided by the manufacturer. RNA concentration was measured in a Qubit 3 Fluorometer with the Qubit™ RNA High Sensitivity (HS) Assay Kit, according to the manufacturer's instructions, RNA integrity was analyzed on a 1% agarose-TAE gel. mRNA sequences were generated using the TruSeq RNA stranded polyA protocol of Useq (Utrecht Sequencing Facility, Utrecht, The Netherlands).
ENA first public
2025-01-21
INSDC center name
Wageningen University & Research
INSDC status
public
Submitter Id
E-MTAB-14756:M. chitwoodi eggs
age
0
broker name
ArrayExpress
collection date
not collected
developmental stage
egg
genotype
Mc31-SMAKT
geographic location (country and/or sea)
not collected
individual
pool of individuals
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-14756:M. chitwoodi eggs
scientific_name
Meloidogyne chitwoodi
sex
undeveloped
strain
NWE-Mc31