RNA-Seq of healthy Sigmodon hispidus heart for de novo transcriptome assembly, replicate 1
In summary, small sections of cotton rat lung (lingular lobe), large intestines (~20 mm colon, flushed with sterile PBS), spleen, kidney, and heart were homogenized using a NextAdvance Bullet Blender with RED RINO tubes containing 3.2 mm stainless steel beads (SSB32) and 2volume of QIAzol Lysis Regent (Qiagen, cat. no. 79306) at max speed for 3 min. RNA was extracted from homogenates using RNeasy Plus Universal Mini kit (Qiagen, cat. No. 73404) via additional QIAzol (total volume 900uL), gDNA eliminator, and chloroform with column DNase digestion as recommend by the manufacturers protocol. RNA quality was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Host rRNA was removed using the NEBNext rRNA Depletion Kit v2 (Cat no: NEB E7400X). The cDNA libraries were prepared using 1 g total RNA from each sample using the NEBNext Ultra II RNA Library Prep Kit for Illumina (Cat no: NEB E7805L) following the manufacturers protocol for intact RNA (RIN>7), AMPure XP Beads for cleanup steps (Beckman, cat. No. A63881), and NEBNext Multiplex Oligos for Illumina (Set 1, cat no: E7600S) for pooling samples. Sequencing was performed using Illumina NovaSeq6000 2150 bp sequencing at the Vanderbilt Technologies for Applied Genomics (VANTAGE) core facility.