Total RNA was extracted from the sorted PD-1+CXCR5+ cells from Tcf7−/−Lef1−/− or control mice, and two biological replicates were obtained for each genotype. cDNA synthesis and amplification were performed using SMARTer Ultra Low Input RNA Kit (Clontech) starting with 10 ng of total RNA per sample following manufacturer’s instructions. cDNA was fragmented with Q800R sonicator (Qsonica) and used as input for NEBNext Ultra DNA Library Preparation Kit (NEB). Libraries were sequenced on Illumina’s HiSeq2000 in single read mode with the read length of 50 nucleotides producing 60–70 million reads per sample. Sequence data in fastq format were generated using CASAVA 1.8.2 processing pipeline from Illumina.
The sequencing quality of RNA-Seq libraries was assessed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, v0.10.1). Because of biased GC content in the 5’ end, the first 12 bases of each read in all 4 samples were trimmed off. RNA-Seq data reproducibility was evaluated by computing Pearson’s correlation of FPKM values for all genes between biological replicates. The Pearson’s correlation coefficient between the two biological replicates was 0.937 for the control samples and 0.986 for the Tcf7−/−Lef1−/− samples, indicating strong reproducibility.
The RNA-Seq libraries were then processed by RSEM (v1.2.19) to estimate expression levels of all genes. The expression level of a gene is expressed as a gene-level FPKM (Fragments Per Kilobase of transcripts per Million mapped reads) value. EBSeq (v1.5.4), as an integral component of RSEM package, was used to identify differentially expressed genes. UCSC genes for mouse mm9 from iGenome (http://support.illumina.com/sequencing/sequencing_software/igenome.html) were used for gene annotation. The RNA-seq data are deposited at the Gene Expression Omnibus under accession number GSE66781.