title: APS4_L2_HER_1
organism: Auanema rhodensis
description: Protocols: Female- and hermaphrodite-fated L2 larvae were isolated by using synchronized progeny populations generated by hermaphrodite mothers. Briefly, dauers (fated to become hermaphrodites) were isolated and allowed to develop into adult hermaphrodites. After ~12 h of egg laying the mothers were removed and the early eggs laid were left to hatch and grow until the L2 stage. During the L2 stage, females and hermaphrodites are distinguishable by their size and coloring: hermaphrodites are smaller, develop slower, are thinner and darker than females. About 200 L2s of each sex were picked and transferred to an Eppendorf tube containing 200 µL of M9 buffer. The nematodes were washed 2-3 times in M9 buffer, allowing them to sink to the bottom of the tube under gravity between each wash. After the final wash, the maximum amount of M9 was removed and 200 µL of Trizol was added to the tube. The tube was placed at -80 °C immediately. For RNA extraction, nematodes were first freeze-cracked in liquid nitrogen (2-3 times). Trizol was added to make up the volume to 500 µL and nematodes were shaken with a few sterile 0.5 mm glass beads on a BeadBeater homogenizer (20 s, 3 times with 30 s intervals). Subsequently, RNA was extracted using a standard chloroform approach and the pellet was dissolved in DEPC treated water and stored at -80 °C. Illumina TruSeq RNA sample Prep kit and protocol was used to construct the mRNA-seq libraries. Briefly, the poly-A containing RNA molecules were purified using poly-T oligo-attached magnetic beads. Following fragmentation of the mRNA, a first strand cDNA synthesis was performed using a reverse transcriptase and random primers, followed by a second strand cDNA synthesis. The cDNA fragments were then end repaired, an 'A' base was added, and finally the adapters were ligated. The resulting products were then purified twice and amplified by PCR to create the final cDNA library.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-02-12T17:04:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-02-11T16:17:59Z
attributes.tag: External Id
attributes.value: SAMEA5332398
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF WARWICK
attributes.tag: INSDC first public
attributes.value: 2019-02-12T17:04:20Z
attributes.tag: INSDC last update
attributes.value: 2019-02-11T16:17:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7667:APS4_L2_HER_1
attributes.tag: age
attributes.value: L2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: L2 larva
attributes.tag: diet
attributes.value: OP50-1
attributes.tag: genotype
attributes.value: APS4
attributes.tag: individual
attributes.value: Manish Parihar
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-7667:APS4_L2_HER_1
attributes.tag: scientific_name
attributes.value: Auanema rhodensis
attributes.tag: sex
attributes.value: hermaphrodite
attributes.tag: strain
attributes.value: APS4