title: GSM4684593: DM_A.AACGCATTT; Mus musculus; RNA-Seq
library_construction_protocol: Mice were aged to 18 months of age in a SPF facility. Upon tissue harvest, mice were euthanized and perfused with 25 ml of cold PBS. The spleen was dissected and splenocytes isolated by using mechanical dissociation. The cell suspension was filtered through a 70 um filter to remove debris. Viable splenic CD3e+CD8a+CD44-PD1-, CD3e+CD8a+CD44+PD1- and CD3e+CD8a+PD1+ cells were sorted using BD Aria II cell sorter, and pelleted at 400 x g 10 min at +4 C. The cell pellet was resuspended in PBS + 0.04% BSA at 1000 cells/uL. mRNA was isolated directly from cell pellets with RNeasy Micro Kit (QIAGEN). mRNA was isolated directly from cell pellets with RNeasy Micro Kit (QIAGEN) and cDNA synthesized using custom oligo-dT primer with a barcode and adaptor-linker sequence (CCTACACGACGCTCTTCCGATCT-XXXXXXXX-T15). Samples were pooled based on Actb qPCR values, RNA-DNA hybrids degraded with acid-alkali treatment and a second sequencing linker (AGATCGGAAGAGCACACGTCTG) was ligated with T4 ligase (NEB). After SPRI-beads clean-up (Agencourt AMPure XP, BeckmanCoulter). the mixture was PCR-enriched (12 cycles) and SPRI-purified yielding final strand-specific RNA-seq libraries.
attributes.tag: GEO Accession
attributes.value: GSM4684593