GSM7060016: FHet.KO.M327fs.GFPp.CD25neg.3; Mus musculus; RNA-Seq
Mouse CD4+ regulatory Tregs in 6-10 weeks old Foxp3 missense mutant mice hemizygous male and heterozygous female were isolated from spleen by flow cytometry, as DAPI–CD19-TCRβ+CD4+CD25+. 1,000 cells were double-sorted directly into lysis buffer for RNA-seq (Smart-seq2)  library preparation following the standard ImmGen low-input protocol (www.immgen.org). total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3) and converted to cDNA via reverse transcription. First-strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were then PCR amplified for 18 cycles using barcoded primers such that each sample carried a specific combination of eight-base Illumina P5 and P7 barcodes and were pooled together prior to Smart sequencing. Smart-seq paired-end sequencing was performed on an Illumina NextSeq500 using 2 x 38bp reads with no further trimming.