title: GSM4038084: T lymphocytes, Sample23_RPMI_Day5; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood mononuclear cells were freshly isolated from blood from healthy human donors of both sexes ranging from 25-60 years old. Naïve T cells were enriched via a negative selection kit (Miltenyi Biotech, 130-097-095) and either used immediately or stored in liquid nitrogen in a 9:1 mixture of FBS: dimethyl sulfoxide. For short-term activation assays, 96 well Nunc Maxisorp plates were coated with 1 to 10 ug/ml of anti-CD3/anti-CD28 antibodies in PBS for 2 to 4 hours at 37°C. Plates were then washed twice with PBS and enriched naïve T cells were added in the indicated media formulations. Levels of activation markers were measured by flow cytometry 16 to 24 hours later. For proliferation assays, cells were stained with Celltrace Violet and then activated as above. After three days, cells were transferred to a new plate with fresh media and analyzed by flow cytometry after another two days. We used 100 ng – 1 ug of total RNA as the input to an mRNA capture with oligo-dT coated magnetic beads using Illumina TruSeq protocol. The mRNA is fragmented, and then a random-primed cDNA synthesis is performed. The resulting double-strand cDNA is used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification and then quantitated by qPCR followed by cluster generation and sequencing