High sensitivity rna screentape kit

Laser-capture microdissection was performed as described in [14] (link). Areas for isolation were defined by a nationally-certified veterinary pathologist, and comprised on one side neoplastic epithelial tissues including all levels of differentiation present and excluding areas with obvious clusters of intraepithelial leukocytes (mainly consisting of neutrophils); on the other side they comprised adjacent, non-neoplastic epithelium. RNA from LCM-isolates was extracted as previously described [15] (link). RNA abundance and quality were analyzed using the 4200 Tape Station Software using the High Sensitivity RNA ScreenTape kit (Agilent Technologies), as detailed in Supplementary Table 4.

Total RNA was extracted from FACS-sorted T. bryosalmonae from brown trout and rainbow trout using RNeasy UCP Micro Kit (Qiagen) along with an on-column DNase digestion step following the manufacturer’s protocol. RNA integrity was assessed on the 4200 TapeStation with the High Sensitivity RNA ScreenTape Kit (Agilent, Santa Clara, CA, USA). Only samples with RNA integrity numbers > 8.0 were used for further analysis.
Library preparation was done with 60 ng total RNA input using the Poly(A) RNA Selection Kit V1.5 and the CORALL mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturers protocol. Six cDNA libraries (3 each for sorted parasite samples from brown trout and rainbow trout) were prepared. Library quality control was done with the High Sensitivity D1000 ScreenTape Kit on the 4200 TapeStation (Agilent). Libraries were sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA, USA) implementing 150-bp paired-end reads. Sequencing was done by the NGS unit of the Vienna Biocenter Core Facilities (VBCF, Vienna, Austria).

Extraction of RNA was performed immediately after microdissection using the Covaris^® truXTRAC FFPE RNA kit according to the manufacturer’s protocol with following adjustments. A sterile blade was used to peel off the thermoplastic film from the LCM cap and transfer the tissue isolated by LCM into glass vials for sonication. Sonication of samples was performed with the E220 focused ultrasonicator (Covaris^®). After reverse crosslinking at 80 °C, the soluble fraction was transferred into clean eppendorf tubes and DNAse treated without prior centrifugation, as due to the absence of paraffin, the samples did not contain any solids that could be precipitated. RNA was eluted from the spin columns using 30 µl of elution buffer prewarmed to 70 °C to increase RNA yield. A second elution into fresh collection tubes was performed using 20–30 µl prewarmed elution buffer using the identical elution protocol. The eluate was aliquoted before analysis and stored at −80 °C. RNA abundance and quality was analysed using the 4200 or 2200 Tape Station Software using the High Sensitivity RNA ScreenTape kit (Agilent Technologies), according to the manufacturer’s protocol.