Computational protocol: Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

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Protocol publication

[…] At the fourth passage post-inoculation, cells were collected and lysed for detection of PrPSc using the Herd-Check CWD Ag Test (IDEXX) as previously described []. The corrected optical density (OD) values (OD450–OD620) of each experiment were each divided by that experiment’s kit-provided positive control to normalize for ELISA plate-to-plate variation; thus, the combined experiment results in are reported as “normalized units”. The kit-provided cut-off value (which is the average of three kit-provided negative controls + 0.18) was used as threshold to determine accumulation of PrPSc. In addition, the corrected OD values of lysates from scrapie-inoculated cells from clone 438 were compared to those of clone 439 using an unpaired t-test with a level of significance of P < 0.01. Also, the corrected OD values of lysates from scrapie-inoculated cells were compared to those of mock-inoculated cells from each clone using a paired t-test with a level of significance of P < 0.01. Statistical analyses were done using JMP Pro 11.2.0. [...] The CLC Genomics Workbench (CLC Bio.) was used to process RNA-Seq data. Mapping parameters were adjusted to map a maximum number of reads to the reference Ovis aries Oar_v3.1 []. The distribution of the expression values for all samples was analyzed and compared. Normalization by quantiles was applied to adjust the distributions for further comparison, as previously described []. Fold changes with respect to RPKM (Reads Per Kilobase per Million mapped reads) values were calculated. Baggerley’s statistical test on proportions was applied to evaluate significance of fold changes []. Bonferroni correction was used to minimize the occurrence of false positives. Comparisons of replicates were performed in order to account for variation of transcription values within each experimental group. Genes with Bonferroni corrected P values below 0.05 were selected for further evaluation. […]

Pipeline specifications

Software tools JMP Pro, CLC Genomics Workbench
Databases Oar
Applications Miscellaneous, RNA-seq analysis
Organisms Ovis aries
Diseases Infection
Chemicals Tretinoin