Illumina HiSeq 4000 paired end sequencing; RNA-Seq of mouse CD4+ T cells to analyse CD4+ TERM cells
RNA-Seq of mouse CD4+ T cells to analyse CD4+ TERM cells
500 cells were sorted as CD4+CD44lowNKG2D- naive T cells or CD4+CD44hiNKG2D- memory T cells or CD4+CD44hiNKG2D+ TERM cells from MACS purified splenic mouse CD4+ T cells Physical dissociation of spleens using metal grids, followed by MACS using the Biolegend negative selection kit for mouse CD4+ T cells straight ex vivo control mice were uninfected SPF mice, memory mice were analysed 45 days after LCMV infection (200ffu LCMW WE i.v.) none applied modified SMART-Seq2:Agencourt RNAClean XP paramagnetic beads (Beckman Coulter) were used in combination with a DynaMag-96 side skirted magnet (Thermo Fisher). cDNA was generated with SuperScript II Reverse Transcriptase Kit (Thermo Fisher) and amplified with HiFi HotStart PCR Mix (KAPA Biosystems). For DNA clean-up Agencourt AMPure XP beads were used (Beckman Coulter) as above.  Nextera XT DNA sample preparation and index kits (Illumina) were used for preparation of libraries