RNA-Seq of O. limacina amoebocytes Rep1
O. limacina specimens were placed individually in 24-well plates with 200 L of 0.22 m filtered seawater. Coelomic fluid was obtained by dissecting the body wall with microsurgical scissors. Amoebocytes and rod-bearing cells were separated manually based on adhesion: amoebocytes adhered to the well surface within 10 minutes, while rod-bearing cells remained suspended and were collected using a Pasteur pipette under light microscopy. Amoebocytes were lysed directly in wells with TRIzol; rod-bearing cells were pelleted (100 g, 5 min), washed, and lysed separately. Lysates from 38 individuals were pooled into three biological replicates per cell type. All procedures were performed at 4C. Total RNA was extracted using TRIzol, treated with DNase I (Thermo), and purified with the CleanRNA Standard kit (Evrogen). RNA quality was assessed via agarose gel and Bioanalyzer 2100 (Agilent). PolyA-enriched RNA was used for cDNA library preparation with TruSeq2 chemistry and sequenced on Illumina NovaSeq6000 (150 bp PE, 37 million reads/sample). Reads were converted to FASTQ using bcl2fastq2 (Illumina).