title: Illumina HiSeq 2500 paired end sequencing; RNA-Seq of human regulatory T cells and effector T cells cultured at standard 37°C temperature and in mild hypothermia (33°C).
design: RNA-Seq of human regulatory T cells and effector T cells cultured at standard 37°C temperature and in mild hypothermia (33°C).
library_construction_protocol: Tregs and CD4+ Teffs were isolated from buffy coats obtained from 3 volunteer blood donors. Treg were cultured at 33°C (mild hypothermia) and were expanded in penicillin (100 U/ml) and streptomycin (100 mg/ml) containing SCGM medium (CellGro, CellGenix, Germany) supplemented with 10% heat inactivated human AB serum, IL-2 (2×103 U/mL, Aldesleukin, Chiron, USA) and beads coated with anti-CD3 and anti-CD28 antibodies (Invitrogen, USA) in a 1:0.6 cell:bead ratio. Beads were added to the cultures at day 0 and 7. Total RNA was isolated from Tregs and Teffs expanded for 14 days in previously described conditions using RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by quality and quantity assessment using 2100 Bioanalyzer (Agilent, Santa Clara, USA). RNA with a RNA Integrity Number (RIN) of 7.0 or above was used for sequencing library preparation. SureSelect Strand Specific mRNA library kit (Agilent, Santa Clara, USA) starting with two rounds of polyA selection was used followed by the standard procedure. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Library Quantification Kit for Illumina (Kapa Biosystems, Woburn, USA) prior to cluster generation.
attributes.tag: Experimental Factor: mild hypothermia (33C)
attributes.value: growth condition
attributes.tag: Experimental Factor: regulatory T cell
attributes.value: cell type