RNA-Seq of Mus Musculus: young male/female spleen
Total RNA was extracted using RNaeasy Kit on QIAcube (Qiagen) from stimulated nave CD8+ T cells for 0 or 24 hours with anti-CD3/28 antibodies which were FACS-sorted from Kmt2dWT or Kmt2d-/- mice. RNA samples were processed following the protocol for the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Poly (A) tailed RNA was purified from 1?g of total RNA, fragmented, and reverse-transcribed into cDNAs. Double strand cDNAs were adenylated at the 3 ends and individually indexed, followed by limited-cycle (15) amplification. Paired-end sequencing (100  2 cycles) of multiplexed mRNA samples per lane was carried out on an Illumina HiSeq 2500 sequencer. Hierarchical clustering analyses were performed with the Cluster software version 3.0 developed by Michael Eisen of Berkeley Lab and the results were compiled using Java Tree View (21). The GO enrichment analysis and KEGG pathway analysis were performed using DAVID software version 6.8 (http://david.abcc.nciferf.gov/) and p < 0.05 was considered to reflect statistical significance.