Computational protocol: SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells

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Protocol publication

[…] Reads were quality-checked with FastQC. Low-quality ends were clipped (3 bases from the start, 10 bases from the end). Trimmed reads were aligned to the reference genome and transcriptome (FASTA and GIFF files, respectively, downloaded from the UCSC mm10 database) with TopHat version 2.0.6. TopHat was run with the following options: mate inner distance set to 30, the corresponding SD to 100, and maximum 10 multi-hits were allowed in the alignment. Reads that did not align at the first attempt were split into 25-base-long sections on which a second attempt of alignment was performed. In B6.129-PrnpZrchI/ZrchI pMΦs, all reads mapping to Prnp between BstEII and EcoRI restriction sites in exon 3 and in the 3′ UTR, respectively, possibly representing a fused mRNA containing neo and residual Prnp, reported in the brain of these mice (), were excluded from the analysis. Polymorphisms were detected using GATK version 2.1.8 using the following options: baq Gap open penalty (whole-genome analysis) set to 30; minimum consensus coverage to genotype indels set to 8 (default: 5); minimum base quality score and minimum variants phred score set to 15. Variants were annotated using snpEFF version 3.0, and distribution of the reads across genomic isoform expression was quantified using RSEM (). Cufflinks version 2.0.2 and differentially expressed genes listed with its utility Cuffdiff using default options were used.All remaining data and statistical analyses, formatting, and picture generating were produced via in-house R-scripts (R version 2.15.2). The SIFT algorithm with default settings was used to predict the impact of a nonsynonymous SNP in Mertk (). […]

Pipeline specifications

Software tools FastQC, TopHat, GATK, SnpEff, RSEM, Cufflinks, SIFT
Application RNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Infection