Phusion polymerase

mRNA was extracted from Melilotus albus and Melilotus siculus using a Qiagen Oligotex mRNA mini kit. Fragmentation of mRNA was done using an Ambion fragmentation buffer. Construction of the cDNA library was based on the Illumina protocol. First strand cDNA synthesis was done using Random Hexamer Primers (Invitrogen) and second strand synthesized using a DNA Polymerase 1 (Promega). End repair was carried out to create uniform blunt ends (Epicentre End-IT repair kit). Unique 4 bp adaptors (Illumina) were added so that the libraries could be pooled for sequencing. An ‘A’ base was added using a Klenow enzyme (3 ^′ to 5 ^′ exo minus, NEB) and adaptor ligation was performed using Epicentre Fast-Link DNA ligation kit. The cDNA template was run on a 2% agarose gel at 120 V for 60 minutes and fragments of approximately 200–500 bp were removed and purified (Zymo gel purification kit). The purified cDNA template was PCR enriched using the Illumina primers and a Phusion polymerase (NEB). The library was quantified using an Invitrogen Qubit fluorometer. Libraries were sequenced on an Illumina Genome Analyzer II under normal conditions and conditions associated with salt tolerance and/or waterlogging tolerance as single-end 100 bp reads, which were trimmed to 71 bp.

Four equal pools (5 individuals) of total RNA were obtained from the PL7, PL10, PL13 and PL16 prawns. The samples for microRNA transcriptome analysis were prepared using a Truseq^TM Small RNA Sample Prep Kit (Illumina, San Diego, USA). Total RNA was ligated with proprietary 5′ and 3′ adapter. Adapter-ligated small RNA was then reverse transcribed to create cDNA constructs using Superscript reverse transcriptase (Invitrogen, CA, USA). The cDNA constructs were subsequently amplied by 15 cycles of PCR using Illumina small RNA primer set and Phusion polymerase (New England Lab, USA), and purified on 6% Novex TBE polyacrylamide gel. Sequencing cycle number is 50 and the reads length is 50 nt with single end sequencing pattern.

In all, 10¹⁰ copies of the plasmid preparation were used for amplification of a 229-bp barcode-containing fragment in a 40-cycle PCR reaction using the Multiplex PCR Plus Kit (Qiagen) according to manufacturer’s protocol, with 57°C as annealing temperature (primers used: BC-PCR-FW and BC-PCR-RV_neu, see Supplementary Table S1, all primers were from Eurofins MWG Operon). PCR products were purified with Agencourt AMPure XP-beads (Beckman Coulter). To attach Illumina adaptors to the PCR products, a tailing PCR was performed with 25 cycles using the Multiplex PCR Plus Kit with 40°C as annealing temperature (primers: Ill1-Tail12 and Ill2_Tail-complete). PCR products were purified with Agencourt AMPure XP-beads afterward. Two microliters of PCR fragments was used for final construction of the indexed Illumina sequencing libraries. A tailed PCR using Illumina indexing primers was performed in 10 µl containing 1× Phusion High Fidelity Mix (NEB), 0.4 U Phusion polymerase (NEB), 5 pmol of a universal primer (P34), an indexing primer and 0.1 pmol of a bridging oligonucleotide. Sequencing was performed on a HiSeq 2000 system (Illumina).