2100 bioanalyzer

Total RNA samples were normalized to the amount of RNA isolated from the corresponding IP sample. rRNA was then depleted from the RNA samples using the Ribominus™ Eukaryote Kit for RNA-Seq (Life Technologies). Depleted samples were ethanol precipitated, washed twice with 70% ethanol and resuspended in 10 μl DEPC water. rRNA depletion was checked on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using a RNA nanochip and the remaining RNA stored at -80°C.
Sequencing libraries were generated using the whole Transcriptome Library Preparation protocol provided with the SOLiD® Total RNA-Seq Kit (Life Technologies). Briefly, rRNA depleted samples were fragmented using RNase III, and subsequently cleaned up using the RiboMinus™ Concentration Modules (Life Technologies). Fragmentation was assessed on a 2100 Bioanalyzer (Agilent Technologies) using the RNA picochip. Fragmented RNAs were reverse transcribed and size selected on a denaturing polyacrylamide gel selecting for 150 to 250 nucleotide cDNA. cDNA was then amplified and barcoded with SOLiD™ RNA Barcoding Kit. Samples were then purified using PureLink™ PCR Micro Kit (Life Technologies) and assessed on a 2100 Bioanalyzer (Agilent Technologies) using the High Sensitivity DNA chip. Samples were deposited on slides, and sequenced using the SOLiD v4 sequencing system (Life Technologies).

Circulating cell-free DNA was extracted by the QIAamp circulating nucleic acid kit from plasma samples. Quantity and quality of the purified cfDNA were checked using Qubit fluorimeter and Bioanalyzer 2100. For samples with severe genomic contamination from peripheral blood cells, a bead-based size selection was performed to remove large genomic fragments. Five to 30 ng of extracted cfDNA were subjected for library construction including end-repair dA-tailing and adapter ligation. Ligated library fragments with appropriate adapters were amplified via PCR. The amplified DNA libraries were then further checked using Bioanalyzer 2100 and samples with sufficient yield are proceeded to hybrid capture.
The 600-gene PredicineATLAS^TM panel with Biotin labelled DNA probes was used for target enrichment. In brief, the library was hybridized overnight with Predicine NGS panel and paramagnetic beads. The unbound fragments were washed away, and the enriched fragments were amplified via PCR amplifications. Similarly as library preparation, the purified product was checked on Bioanalyzer 2100 and then loaded into Illumina NovaSeq 6000 for NGS sequencing with paired-end 2x150bp sequencing kits.

Total RNA enriched for small RNA, including miRNA was isolated from SCAT samples using the Qiagen miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendation. The quality and quantity of small RNA were assessed using an Agilent Small RNA kit on a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Samples were then pooled (4 pools with 4 individuals per group and day). Pooling of samples was performed at the RNA level by mixing randomly selected RNA samples extracted from independent biological samples from the same group and equal amounts of RNA from each vole, before library preparation.
Libraries were prepared with NEXTFLEX Small RNA-seq v3 Kits with UDIs for Illumina (PerkinElmer) according to the manufacturer’s recommendations. The quality of the libraries was assessed using the Highly Sensitive DNA assay and a 2100 Bioanalyzer (Agilent). A total of 12 multiplexed libraries were parallel sequenced for single-end of 50 bp using the rapid-run mode of the Illumina HiSeq 2500 system by the sequencing facility of the Genome Biology Institute, FBN, Dummerstorf, Germany.