WT_4weeks_LO3
Two experimental designs were used for sample collection. In the first experiment, we used juveniles from different clutches (L108 and L117) laid by the same female, and we equally pooled the two clutches for the collection. In the second experiment, we used juveniles from one large clutch laid by 1-3 females.Squid were collected at 11 hours post dawn, stabilized overnight at 4°C in RNAlater (Ambion), and frozen at -80°C until dissection. RNA from a pool of 6-8  light organs per experimental condition was extracted using the RNeasy kit (Qiagen), with the following modifications to the manufacturer’s instructions. RNA was then treated for potential DNA contamination using the Turbo DNase kit (Ambion). The quality of RNA extracts was checked using a 2100 Bioanalyzer (Agilent) and its quantity measured using the Qubit technology (Life Technology). Libraries were prepared and sequenced by the UW-Biotechnology Center, starting from 1 µg of total RNA for the ‘Apo’ and ‘?lux’ conditions and from 1.3 µg of total RNA for the ‘WT’ condition, to correct for the under-estimation of the squid RNA quantity because of the presence of bacterial RNA in the ‘WT’ samples. All samples were prepared simultaneously using the TruSeq Illumina technology [TruSeq mRNA protocol Rev.A with a 6-min fragmentation step and a ligation step using 18 specific adaptors (1/condition)]. Libraries were validated on a Biotek Synergy2 Plate Reader PicoGreen before being sequenced across 6 lanes (18 samples/lane) on a HiSeq2000 (1x100bp).