RNA sequencing
Total RNA was isolated from Fut1 KO and C57BL/6 WT splenocytes, and RNA quality was assessed using Agilent 2100 bioanalyzer with the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands). A total of 500 ng of RNA from each group was used for RNA sequencing.
For RNA sequencing, library construction was performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturer’s instructions. The completed library was purified to remove PCR components. High-throughput sequencing was carried out as single-end 75 sequencing using the NextSeq 500 platform (Illumina, San Diego, CA).
For data analysis, the QuantSeq 3’ mRNA-Seq reads were aligned using Bowtie2. Bowtie2 indices were either generated from genome assembly sequence or the representative transcript sequences for aligning to the genome and transcriptome. The resulting alignment file was used for transcript assembly, abundance estimation and detection of differential gene expression. Differentially expressed genes (DEGs) were determined based on counts from unique and multiple alignments using coverage in Bedtools (11). The Read Count data were processed using the quantile normalization method with EdgeR within R using Bioconductor (12). Gene classification was performed based on searches conducted by DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov/).