time course: 2nd sensitization treatment: tick infestation
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the skin biopsies using the RNeasy Plus kit (Quick RNA miniprep plus kit from Zymo, USA).
Label
NanoString
Label protocol
none provided
Hybridization protocol
Hybridization was performed per the manufacturer’s instructions for Gene Expression Panels with Panel Plus/CodeSet with CodeSet Plus. Briefly, Hybridization was set up with 5 mcL of sample (100 ng of RNA), which was added to 10 mcL of hybridization master mix (5 mcL of hybridization buffer + 3 mcL of Reporter CodeSet + 2 mcL of Reporter Plus) + 3 mcL of Capture Maser Mix. The total hybridization volume was 18uL per sample. Each cartridge was hybridized for 18 hours on a thermal cycler set to 65℃ with a lid heated to 70℃.
Scan protocol
Cartridges were then analyzed on the nCounter Pro Analysis System
Data processing
Nanostring Data was analyzed by ROSALIND (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step. Normalization, fold changes, and p-values were calculated using criteria provided by Nanostring.
