GSM5454465: Pirc rat with no tumor not fed with spinach, PCN2_RNAseq; Rattus norvegicus; RNA-Seq
The colonic tissues were snap frozen with liquid nitrogen after the rat necropsy and each tissue sample was lysed in 700 ul Qiazol lysis reagent with tissue homogenizer. Total RNA was isolated from up to 50mg colonic tissue using Qiagen miRNeasy mini kit, according to manufacture's insturctions to cover both mRNA and miRNA. The isolated RNA concentration was measured by a Qubit 4 Fluorometer with the Qubit™ RNA HS Assay Kit  (Thermo Fisher Scientific). For RNA library preparation, NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and NEBNext® UltraTM Directional RNA Library Prep Kit (NEB, E7420) were performed with 3 mg RNA input as the starting material. For small RNA library preparation, NEBNext® Small RNA Library Prep Set for Illumina® (NEB, E7300) were performed with 1 mg RNA input as the starting material. The RNA went through 3' ligation, primer hybridization, 5' ligation, first strand synthesis, PCR amplification and size selection (MinElute Gel Extraction Kit, Qiagen). During library construction, each sample was tagged with a specific index at the PCR amplification step. The library concentration was measured by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the size of the library was assessed by 2% agarose gel with 2 ng library material. Samples were diluted to 1 pmol for further library pooling. Before loading the pooled library into the NextSeq 550/500 system (Illumina), 1 nM library was denatured and diluted according to NextSeq  System Denature and Dilute Libraries Guide (Illumina). Stranded RNA sequencing was conducted by paired-end sequencing with 75-bp read length and small RNA sequencing was conducted by single-end sequencing with 50 bp read length following the manufacturer's protocols.