title: Illumina HiSeq 2500 sequencing; Expansion of MHC-E restricted CD8+ T cell responses in blood and intestine during non-pathogenic SIV infection
design: Expansion of MHC-E restricted CD8+ T cell responses in blood and intestine during non-pathogenic SIV infection
library_construction_protocol: Three distinct CD8+ T cell subpopulations were sorted from blood of five chronically SIVagm-infected AGM: the MHC-E-restricted CD8+T cells, and the memory and effector conventional CD8+T cells.  RNA was isolated from the sorted cells using the RNeasy® Mini Kit (205113, Qiagen). RNA integrity was verified with the Agilent Bioanalyzer. DNAse-treated RNA molecules were treated for library preparation using the Truseq Stranded mRNA sample preparation kit (Illumina, San Diego, California) according to manufacturer's instructions.  The fragmented RNA samples were randomly primed for reverse transcription followed by second-strand synthesis to create double-stranded cDNA fragments. No end repair step was necessary. An adenine was added to the 3'-end and specific Illumina adapters were ligated. Ligation products were submitted to PCR amplification. The obtained oriented libraries were controlled by Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified by spectrofluorimetry (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen).