Smarter v4 ultra low input rna kit

Plankton sampled in November 2015 and November 2016 in the Little Bay of Toulon, France were preserved in 70% ethanol and stored at $-20$ °C. The copepods were isolated under the stereomicroscope as previously described. We selected O. nana individuals from five different development stages: five pairs of egg sacs, four nauplii (larvae), four copepodites (juveniles), four adult females, and four adult males. All individuals were isolated from the November 2015 sampling, except for the eggs. Each individual was isolated, then crushed with a tissue grinder (Axygen, Corning, Arizona, MA, USA) into a 1.5 mL Eppendorf tube. Total mRNAs were extracted with the NucleoSpin RNA XS kit (Macherey-Nagel, GmbH & Co., Duren, Germany) following the manufacturer’s instructions and then quantified on a Qubit 2.0 with the RNA HS Assay kit (ThermoFisher Scientific, Waltham, MA, USA); quality was assessed on a Bioanalyzer 2100 with the RNA 6000 Pico Assay kit (Agilent, Gilent Techlonoly, Santa Clara, CA, USA). cDNAs were constructed using the SMARTer v4 Ultra low Input RNA kit (Takara, San Jose, CA, USA). After cDNA shearing using a Covaris E210 instrument, Illumina libraries were constructed using the NEBNext Ultra II kit (New England Biolabs, MA, USA) and sequenced on an Illumina HiSeq2500. A minimum of 9.7e6 reads pairs were produced from each individual (Supplementary Notes S1).

cDNA from each sample was generated from 10ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech). The cDNA was amplified by 8 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent). Sequence libraries were produced from 150pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX. Single-end sequencing data were generated on an Illumina HiSeq 2500, at a depth of 30 million reads per sample, with read length 50bp.