GSM2973430: CD4+Foxp3+CD25- uTregs NP-1; Mus musculus; RNA-Seq
Uterine and uDLN Tregs and uterine CD4conv were isolated by cell sorting using FACS ARIA (BD Biosciences) after enrichment in CD4+ cells was performed using the EasySep Mouse APC Positive Selection Kit (Stemcell 18452). Groups of 20 mice per non-pregnant sample and 3 to 8 mice per pregnant sample were made and 4 samples were collected per cell type. RNA was purified with the RNAqueous®-Micro Total RNA Isolation Kit (Invitrogen AM1931). cDNA was prepared with the SMART-Seq v4 Ultra Low Input RNA Kit. Indexed paired-end libraries were done with the Nextera® XT Library Prep Kit and the Nextera XT Index Kit. The quality control was done in Tapestation and Quantifluor. Illumina sequencing of the 16 samples in 2x150, 50 million reads/sample was done with the NextSeq 500 High Output Kit v2 (300 cycles) sequencer (Illumina Inc) generating paired end reads of approximately 2 x 150 bp in length. The samples were sequenced in two batches; CD4conv and uTreg E6-E8 samples in the first batch, and uDLN Treg NP and uTreg NP samples in the second batch.