Illumina HiSeq 2500 paired end sequencing; RNAseq of Tregs before and after treatment with Multipep
RNAseq of Tregs before and after treatment with Multipep
Cryopreserved PBMC samples (10^6 cells) from baseline (1 or 2 weeks before first treatment) and 24 weeks post treatment timepoint were thawed from 10 subjects  and stained first using live/dead violet then surface stained using CD14 CD3 CD4 CD25 CD127 antibodies. A minimum of 40,000 CD14- CD3+ CD4+ CD25+ CD127lo Tregs (average = 81,600, max = 158,300) were sorted using BD FACSAria III and frozen at -80C in minimal PBS prior to DNA/RNA extraction.  DNA/RNA was extracted in parallel with the AllPrep micro kit (Qiagen)  RNAseq libraries prepared from 2/3 of extracted RNA (91-140 ng) with SMART-Seq v4 and NuGen Ovation Ultralow