GSE129472
Gene expression profiles of Foxp3RFP (WT), Foxp3?EGFPiCre (Foxp3-deficient) and Foxp3?EGFPiCreRictor?/? (Foxp3-deficient and Rictor deficient) Treg cells.
Comparaison of the transcriptomes of WT and Foxp3-deficient regulatory T cells from the spleen of  females that carried one mutant Foxp3?EGFPiCre allele and a second competent Foxp3 allele (Foxp3RFP) that also directed the expression of the red fluorescent protein (RFP) . We then extended these studies to elucidate transcriptional pathways altered upon Rictor deletion in ?Treg cells that may contribute to their improved regulatory function by analyzing the transcriptome of ?Treg cells isolated from Foxp3?EGFPiCre/+Rictor?/? females. Overall design: Splenic Treg (CD4+RFP+ or CD4+YFP+) cells were double-sorted from 4 weeks old female Foxp3?EGFPiCre/RFPR26YFP and Foxp3?EGFPiCre/+Rictor?/?R26YFP mice. (n=4 per group). Cells were collected directly into TRIzol (Invitrogen). Total RNA was extracted and converted into double-stranded DNA (dsDNA), using SMART-Seq v4 Ultra Low Input RNA kit (Clontech). dsDNA was then fragmented to 200- to 300-bp-sized fragments, using M220 Focused-ultrasonicator (Covaris), and these were used for the construction of libraries for Illumina sequencing, using the KAPA Hyper Prep Kit (Kapa Biosystems). Libraries were then quantified using Qubit dsDNA HS (high-sensitivity) Assay Kit on Agilent High Sensitivity DNA Bioanalyzer. RNA sequencing data was demultiplexed by using perfect matches to indices and was quality-inspected using FastQC. The sequencing data was aligned to the mm10 build (Gencode annotation) of the mouse genome using STAR, and counts were quantified using HTSeq-Counts.