RNA-seq of hydrovascular fluid coelomocytes in control Holothuria forskali
RNA extractions were carried out using the kit Quiagen RNeasy according to the manufacturer's instructions. The concentration and purity of extracted RNA were determined using a nanodrop spectrophotometer (Denovix DS11), and the RNA integrity value (RIN) was assessed using the Agilent 2100 bioanalyzer (Agilent RNA 6000 Nano kit). Only the three sample showing the best RNA quality of HF and PF were retained for RNA-seq. cDNA library preparation and sequencing were carried out by the Beijing Genomics Institute (BGI, Hong Kong). Briefly, the cDNA libraries were assembled as follows: mRNAs were isolated from total RNA using the oligo(dT) method; purified mRNAs were fragmented and reverse transcribed into the first cDNA strand, prior to synthesis of the second cDNA strand; Double-stranded cDNA fragments were end-repaired, 3'-adenylated and ligated to sequencing adapters; cDNA fragments of appropriate size were selected and enriched by PCR; PCR products were heat-denatured and single-stranded DNA was cyclized by oligo splint and DNA ligase. The libraries were then sequenced on the BGISEQ-500 platform.