GSE173805
Type 1 calreticulin mutations activate the IRE1a-XBP1 pathway of the unfolded protein response to drive MPN
Experimental data indicates unique activation of the unfolded response in calreticulin (CALR) mutant myeloproliferative neoplasms (MPN). To dissect UPR activation in an MPN, calreticulin-mutant context at the transcriptional level, RNA-seq was performed. Cell lines were generated with the addition of a plasmid containing the thrombopoietin receptor (MPL) in addition to a CALR variant: either wild-type calreticulin, calreticulin with a deletion of fifty-two base pairs/type I mutant, or calreticulin with an insertion of five base pairs/type II mutant. Cells were cultured in growth media containing 10% FBS RPMI with penicillin/streptomycin addition in a 5% CO2 incubator. Cells were washed three times with PBS followed by media replacement using growth media with or without IL3 addition. In unstarved conditions, cells were supplied with the cytokine IL3 at a 1:10,000 dilution for a final concentration of 2 ng/mL; in starved conditions, cells were depleted of the IL3 cytokine addition for twenty-four hours. RNA-seq was performed on both unstarved and starved cells in triplicates for each genotype. Additionally, for preliminary data purposes, a cell line was generated with MPL plasmid transduction and with the transduction of a plasmid containing the JAK2 kinase with the V617F mutation to examine MPN activation in a JAK2 mutant, MPN context; these samples (unstarved and starved) were not sequenced in triplicates but with one sample each. Overall design: Triplicates for each calreticulin genotype in conditions of cytokine addition of IL3 (unstarved) or following cytokine absence for 24 hours (starved); only one unstarved and one starved sample of the JAK2VF-MPL cell line was conducted. RNA sample quality check, library construction, and sequencing were performed by the University of Chicago Genomics Facility following standard protocols. All samples were sequenced in two runs by a NovaSeq 6000 sequencer to generate paired-end 100bp reads. For each sample, the raw FASTQ files from two flow cells were combined before downstream processing. RNA-seq data were processed using a local Galaxy 20.05 instance for the following steps. Quality and adapter trimming were performed on the raw sequencing reads using Trim Galore! 0.6.3. The reads were mapped to both the mouse genome (GRCm38.p4 with GENCODE annotation) using RNA STAR 2.7.5b. The resulting mapped reads from each sample were counted by featureCounts 1.6.4 for per gene read counts.