Illumina HiSeq 2500 paired end sequencing; RNA sequencing of CD57+ and CD57- effector memory CD8 T cells in patients with type 1 diabetes
RNA sequencing of CD57+ and CD57- effector memory CD8 T cells in patients with type 1 diabetes
Blood samples were taken from five HLA-A*24+ males with newly diagnosed type 1 diabetes. PBMCs were isolated on density gradients using Lymphoprep (Nycomed) and washed in RPMI supplemented with 2% human AB serum. PBMCs were washed and stained as above and CD57+ and CD57- effector memory CD8 T cells (gated as live CD14-CD16-CD19-CD4-CD3+CD8+CCR7-CD45RA-CD27-) were FACS-sorted using an Aria (BD Biosciences) into BSA supplemented with 10% BSA/5 mM EDTA. RNA was isolated immediately from cell pellets using the PicoPure RNA extraction kit (ThermoFisher). cDNA was generated and amplified using the SMARTseq v4 Ultra Low Input RNA Kit (Clontech) on the contactless Labcyte liquid handling system (Labcyte Inc). 200 ng of amplified cDNA was used from each sample where possible to generate libraries using the Ovation Ultralow Library System V2 kit (NuGEN). In brief, cDNA was fragmented through sonication on Covaris E220 (Covaris Inc), repaired and polished followed by ligation of indexed adapters. Adapter ligated cDNA was pooled before final amplification to add flow cell primers.