GSM5590340: Old Female CD4+ Naïve #2; Mus musculus; RNA-Seq
Lymphocytes were prepared from pooled lymph nodes (axillary, brachial, cervical, inguinal) and splenocytes then purified for CD4+ T cells through negative selection by EasySep CD4+ T cell Isolation Kit (STEMCELL Technologies, Cat#19852). Cell suspensions are then stained and FACS-sorted for either naïve (CD3+CD4+CD25-CD62L+CD44-) or memory (CD3+CD4+CD25-CD62L-CD44+) populations by BD FACSAria Fusion Sorter. RNA was isolated using RNeasy Plus Micro Kit (Qiagen, Cat#74034). Subsequently, 125ng of total RNA was used for the construction of sequencing libraries using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Cat#E7645). RNA libraries were prepared for sequencing according to New England Biolab's instructions accompanying the aforementioned kit with the additional use of AMPure XP beads (Beckman Coulter, Cat# A63880), NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Cat# E7490), and NEBNext Multiplex Oligos for Illumina (NEB, Index Primers Set 1 Cat#E7335 and Index Primers Set 2 Cat# E7500).