GSM7991677: vagina sham 14; Oryctolagus cuniculus; RNA-Seq
Rabbit samples were removed from the freezer, and up to 30 mg of frozen tissue was transferred to a 2 mL tube containing steel beads and 0.5 mL lysis buffer (RLT buffer Qiagen, Valencia CA; product nr. 217684) the tubes were placed in the precooled tissue-lyser LT scaffold (Qiagen, product nr. 85600) and homogenized for 50 sec over two cycles. Each cycle was accompanied by a cooling time on ice for 30 sec. RNA was isolated from the homogenized samples by using the miRNeasy Tissue/cells advance kit (Qiagen, Valencia, CA) by following the manufacturer's instructions. In brief, genomic DNA and proteins were eliminated by using a DNA eliminator spin column, and proteins were eliminated by using a protein precipitating reagent (PPR) and Proteinase K step. The resulting RNA solutions were next applied to a RNeasy Mini Spin Column. RNAs were eluted in 30 µl volume of RNase-Free Water. The RNA concentration and quality were evaluated using Qubit 2.0 Fluorometer and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), Respectively, at Karolinska Institutet, KIGene Core Facility. We processed the RNA samples for sequencing using the Illumina TruSeq Stranded mRNA (polyA) library kit. These fragments underwent sequencing on a NovaSeq 6000 system. We converted Bcl files to FastQ format with the assistance of the CASAVA software. The quality measurement adhered to the Sanger / phred33 / Illumina 1.8+ scale. The National Genomics Infrastructure in Genomics Production Stockholm oversaw the quality assessment, focusing on factors like yield, read quality, and any potential cross-contamination between samples. After demultiplexing and conversion to FastQ format on location, we then uploaded the data to UPPMAX for delivery.