Nebnext ultra 2 kit

On microgram of purified gDNA was used with a QIAamp DNA mini kit, two biological replicates for each condition (QIAGEN, catalog no. 51304). Briefly, for methylated DNA immune precipitation and purification, a MagMeDIP-seq kit was used (Diagenode, code C02010040). First, gDNA was sonicated to obtain a fragment size between 150 and 300 bp, and then it was denaturated to single-stranded DNA and immune-precipitated using an anti-methylcytosine antibody provided by the kit. The next day, immunoprecipitated DNA and input were purified and eluted. Library preparation was performed using the NEBNext Ultra II kit for Illumina (code E7645), following the manufacturer’s instruction. Each library was dual-indexed using NEBNext Multiplex Oligos for Illumina (code E6440) and sequenced at 30 million pair-end depth with Illumina HiSeq 2000.

Libraries for Illumina sequencing were prepared with the directional RNA NEBNext® Ultra™ II kit according to the manufacturer’s instructions. Briefly, samples were enriched in mRNA by selecting 3′ end-poly(A) tails with the magnetic isolation module NEBNext Poly(A). The mRNA was transformed to cDNA, and sequencing adaptors were added prior to sequencing. Samples were dual indexed for demultiplexation after sequencing. A Bioanalyzer Agilent 2100 was used to check the fragment size distribution and concentration (Agilent HS kit). Libraries were quantified with the assay kit Qubit dsDNA HS (Thermo Fisher Scientific) and grouped in equimolar amounts according to Qubit. These groups were sequenced on a NovaSeq PE150 flow cell to achieve a total of 60 gigabases.

A Chicago (RRID:SCR_014941) library was prepared as described in [34 (link)]. Briefly, ∼500 ng high molecular weight gDNA was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5′ overhangs were filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ∼350-bp fragments and sequencing libraries were generated using the NEBNext Ultra II kit with Illumina-compatible indices. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeq X Ten (RRID:SCR_016385) to produce 467 million 2 × 150-bp paired-end reads.

Genomic DNA size and quality was assayed using the NanoDrop and Agilent 4200 TapeStation. Libraries were prepared using 100–500 µg DNA and the NEBNext Ultra II kit with Unique Dual Index primers for Illumina. Enzymatic fragmentation was carried out for 15 min instead of 5. Libraries were amplified between four and six cycles using the NEBNext UDI primers (article E6440). Library size and molarity was determined using the TapeStation system and libraries pooled at a concentration of 2 µM. Paired-end 100 bp sequencing was performed using the NextSeq 2000 and NovaSeq platforms.

To conduct a functional analysis, Mediterranean O. similis transcriptomes were produced. Oithona similis specimens were sampled at the North of the Large Bay of Toulon, France (Lat 43°06’ 02.3” N and Long 05°56’ 53.4” E). Sampling took place in November 2016. The samples were collected from the upper water layers (0–10 m) using zooplankton nets with a mesh of 90µm and 200 µm (0.5 m diameter and 2.5 m length). Samples were preserved in 70% ethanol and stored at −4°C. From the Large Bay of Toulon samples, O. similis individuals were isolated under the stereomicroscope (Nishida, 1985; Rose, 1933). We selected two different development stages: four copepodites (juveniles) and four adult males. Each individual was transferred separately and crushed, with a tissue grinder (Axygen) into a 1.5 ml tube (Eppendorf). Total mRNAs were extracted using the ‘RNA isolation’ protocol from NucleoSpin RNA XS kit (Macherey‐Nagel) and quantified on a Qubit 2.0 with a RNA HS Assay kit (Invitrogen) and on a Bioanalyzer 2100 with a RNA 6000 Pico Assay kit (Agilent). cDNA was constructed using the SMARTer‐Seq v4 Ultra low Input RNA kit (ClonTech). The libraries were built using the NEBNext Ultra II kit for paired‐end sequencing with an Illumina HiSeq2500. After adaptors trimming, only reads with a mean Phred score > 20 were kept.