Screen tape system

The pyloric caecum, liver, and muscle tissues were collected from selected fish samples from the red and white fillet groups. The tissue samples were immediately flash-frozen in liquid nitrogen before transferring to -80^oc for storage. Total RNA was extracted from the tissues using the RNAzol reagent (Molecular Research Center Inc., USA) method following the manufacturer’s instructions. The concentration of RNA was measured by NanoDrop spectrophotometer Gen 5 version 2.09.2 (BioTek Instruments, Inc., USA), and the purity was estimated by the A260:A280 ratio. Gel electrophoresis was used to confirm the integrity of the extracted RNA. RNA samples selected for library preparation had an A260:A280 ratio of 1.8–2.1. RNA integrity was assessed with a ScreenTape® system (Agilent, Santa Clara, CA) and samples with RIN (RNA Integrity Number) of 7 or above were used. A total of ten (10), twenty-three (23), and sixteen (16) individual fish RNA samples were extracted from the pyloric caecum, liver, and muscle, respectively. The RNA were individually sequenced and used for this study.

Messenger RNA from whole-cell lysates was isolated using TRIzol^® reagent (Life Technologies), and libraries were prepared using an Illumina TruSeq Stranded mRNA library prep kit. Individual MSCs were isolated using a pressure-gated microfluidic chip. Single MSCs were processed using the REPLI-g WTA single-cell (Qiagen). The amplified double-stranded cDNA was fragmented using NEB double-stranded DNA fragments. An Agilent screen tape system was used to quantify 100 ng of fragmented DNA for library prep input. A NEBNext Ultra II DNA library prep kit for Illumina Barcoded libraries was used to process 100 ng of fragmented cDNA, and the resulting products were submitted for RNA sequencing by the Loma Linda University Center for Genomics.