GSM9001702: 2.12 Adult day 1; Sigmodon hispidus; RNA-Seq
RNA was extracted and isolated with RNeasy Plus Mini extraction kits (QIAGEN) following manufacturer instructions, with a final elution volume of 30 µL. RNA concentrations, 260/280 ratios, and 260/230 ratios were measured using a NanoDrop One spectrophotometer (Thermo Fisher), ensuring a minimum concentration of 100 ng/µL and 260/280 ratios between 1.8 and 2.2 for all extracted RNA samples. The RNA Integrity Number (RIN) for DNase-treated total RNA were assessed using Agilent BioAnalyzer RNA Nano Kit (Agilent Technologies, Inc., Santa Clara, CA) and the RNA amount was assayed with the Invitrogen Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA). Samples with RIN > 7 were used in mRNA-seq library generation using the QIAseq FastSelect RNA Lib Core Kit (QIAGEN) and the QIAseq Stranded mRNA Enrichment Kit (QIAGEN). Briefly, 100 ng DNase-treated total RNA was used as input for library generation. RNA fragmentation was set for 3 minutes at 94C. 17 PCR cycles were used in final library generation. Library quantification and characterization were assessed with Agilent BioAnalyzer HS DNA Kit and the Invitrogen Qubit DNA HS Assay Kit. Libraries were pooled together with other index – compatible RNA-seq libraries for sequencing on Illumina NovaSeq X Plus 10B (Illumina Technology, Portland, OR) flow cell lane to a minimum depth of 15 - 20 million clusters per sample.