Computational protocol: Human Neutrophils Produce CCL23 in Response to Various TLR Agonists and TNFα

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Protocol publication

[…] Total RNA extracted from neutrophils (50 × 106/condition) was quality checked by Agilent 2100 Bioanalyzer (Agilent Technologies). RNA integrity (RIN) was routinely found to be ≥7.0. RNA-seq libraries were prepared by oligo-dT selection using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina HiSeq 2000. After quality filtering according to the Illumina pipeline, 51-base-pair (bp) reads were aligned to the hg19 assembly (Genome Reference Consortium GRCh37) as well as the human transcriptome reference (UCSC annotation), using TopHat (Trapnell et al., ). We allowed up to two mismatches and specified a mean distance between pairs (–r) of 250 bp. The reference sequence and annotation files were downloaded from iGenomes repository at the following website: Read counts per gene were obtained from the aligned reads using “htseq-count” command from the HTSeq framework version 0.6.1p1 (Anders et al., ), with the following parameters: “–f bam –r name –m union –s no –t exon –i gene id”. Count normalization was performed using the Bioconductor/R packages DESeq2 (Love et al., ). Transcript abundance was indicated as Fragments Per Kilobase of transcript sequence per Million of mapped fragments (FPKM), using the “fpkm” function of DESeq2. […]

Pipeline specifications

Software tools TopHat, HTSeq, DESeq2
Databases iGenomes GRC
Organisms Homo sapiens