RNA-seq from Poecilia reticulata ovary
Total RNA was isolated using the Direct-zol RNA MicroPrep (Zymo research, Irvine, CA, USA) following manufacturer's instruction, including also a DNAse treatment. Total RNA was quantified with NanoDrop ND-1000 (Thermo Scientific) and its quality assessed with 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples had an RNA Integrity Number (RIN) value higher than 8. Finally, equal amounts of RNA from four different females within the same experimental group were pooled together, so that we obtained a total of three pools for each treatment (C_1h, EJ _1h, C_6h, EJ_6h). Tagged RNA-seq libraries were therefore prepared from RNA pools using Agilent's SureSelect Strand Specific RNA Library Preparation Kit (Agilent Technologies), following manufacturer's instruction. Briefly, poly(A) mRNA was purified from 500 ng of total extracted RNA and fragmented using an RNA-seq Fragmentation Mix. First-strand and second-strand cDNA were synthetized and end-repaired. Adenylation of cDNA 3' ends and adaptor ligation were performed. Sixteen cycles of PCR were used to amplify and index the adaptor-ligated cDNA library, and the PCR products were purified and size selected using the SPRIselect reagent kit (Beckman Coulter, Brea, CA, USA). Library concentrations were measured by both a Qubit RNA Assay kit in a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and a PCR-based method by using the NEBNext Library Quant Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Individual libraries were monitored for insert size using the Agilent DNA 1000 assay on the Agilent Bioanalyzer 2100 system (Agilent Technologies). Multiplexed single-end sequencing 50 bp was carried out on an Illumina Hi-Seq 4000 (Fasteris, Geneva, Switzerland).
SUB4499556
6h_C_1