Ribosome profilling and RNA Sequencing of WT and Rasgrp1Anaef mouse CD4+ T cells

To determine what mRNAs are translated at baseline in CD4+ T cells, and whether these are altered in Rasgrp1Anaef CD4+ T cells, which have a point mutation in the Ras GEF Rasgrp1, before the onset of autoimmunity, we performed sequencing of ribosome-protected fragments as well as total RNA from WT and Anaef T cells. We identified a number of target genes that are translated at baseline, many of which have been shown to be downstream of the kinase mTOR. CD4+ T cells from the lymph nodes of mice that are WT/WT or Rasgrp1Anaef/Rasgrp1Anaef were purified by a negative isolation magnetic bead-based kit. 20000 cells were lysed and total RNA was isolated (4 samples per genotpye) and RNA was sequenced (Illumina HiSeq 4000); from the rest of the cells in the pool. samples were pooled 2 by 2 (for 2 total samples per genotype), lysed, and ribosome-associated mRNAs were isolated and sequenced (Illumina HiSeq 4000)