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/).