Rna 6000 pico assay kit

The harvested cell as well as tissue samples were treated by utilizing a miRCURY RNA Isolation Kit (Exiqon, Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer to isolated cellular RNA. Then, the isolated RNA was assayed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Mountain View, CA) in conjunction with an RNA 6000 Pico assay kit (Agilent Technologies, Mountain View, CA) according to the recommended assay methods provided by the assay kit manufacturer to quantify the RNA concentration. In the next step, 1 μg of isolated total RNA was converted into cDNA by making use of a QuantiTect Reverse Transcription assay kit (Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer. Finally, real time quantitative polymerase chain reaction (RT-qPCR) was performed on a BX-384 real time PCR apparatus (Bio-Rad laboratories, Hercules, CA) by making use of a QuantiTect SYBR Green PCR assay kit (Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer to evaluate the relative expression of circFNDC2B, miR-942, miR-510, CD44 mRNA as well as CDH1 mRNA in each sample using the 2^−ΔΔCt approach. The expression of GAPDH in each sample was used as the internal control.

RNA samples were extracted from the leaves of three replicates of control and the 24-h and 10-day cold-stress-treated plants. Total RNA was extracted using a Spectrum Plant Total RNA kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions. Any DNA contamination was removed by DNase I treatment on column (Roche, Basel, Switzerland). The RNA quality tests were performed with the Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA) using an RNA 6000 Pico assay kit (Agilent Technologies, Santa Clara, CA, USA). We pooled RNA from each time and complementary DNA (cDNA) libraries and sequencing in Illumina HiSeq 1000 sequencer was performed by GeneSystems (Valencia, Spain). Two replicates of each sample were sequenced on different lanes in the flow cell.

For the two samples, total RNA was extracted by using a PicoPure RNA isolation kit (Acturus) and subjected to DNase treatment (RNase-free DNA set; Qiagen) according to the manufacturer’s protocol. Next, 10 ng RNA was used for cDNA synthesis and amplification by using an Ovation RNA-seq System V2 kit (NuGen Technologies) following the manufacturer’s protocols with minor modifications. The quality and profile of the RNA and amplified cDNA was assessed by using an RNA 6000 Pico Assay Kit with an Agilent 2100 Bioanalyzer, respectively (Agilent Technologies). The cDNA libraries were then further refined by using the Illumina TruSeq RNA Sample Preparation v2 kit (Illumina) with the low-throughput protocol according to the manufacturer’s instructions. Then samples were paired-end sequenced by using an Illumina HiSeq 2500 platform (Illumina Inc.)¹. The average length of the reads was 125 bp.

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).