Dataset features


Application: RNA-seq analysis
Number of samples: 16
Release date: Jun 12 2017
Last update date: Nov 30 2018
Access: Public
Diseases: Border Disease
Dataset link Plexin B2 and Semaphorin 4C guide T-cell recruitment and function in the germinal center

Experimental Protocol

Plxnb2+/+ or Plxnb2-/- CFP-expressing MD4 B cells were co-transferred together with OT-II T cells into B6 recipients that were subsequently immunized with HEL-OVA subcutaneously. MD4 cells of the 7-AAD-CD19+IgD-GL7hiFashi GC phenotype were FACS-sorted from pooled draining lymph nodes on day 5. To conduct transcriptomic RNA-seq analyses on these cells, a protocol initially developed for single-cell RNA-seq (Tang et al., 2011) was modified to accommodate 400 sorted cells by doubling reaction volumes with extra buffers until the step for second strand DNA synthesis. Cells were directly sorted into the lysis buffer, and reverse transcription was carried out for individual sorts within 20 minutes after isolation to preserve sample integrity. SE-100 sequencing was conducted for all samples on a HiSeq 2500 sequencer (Illumina) at the Tsinghua. Sequence reads were aligned to the Mus musculus reference genome using TopHat2 and assembled by Cufflinks to calculate the FPKM for each transcript. Genes with an average read number of at least 1 were subjected to differential expression analysis by the DESeq2 software (Bioconductor) with a call threshold set at padj<0.1.










Hai Qi