GSM8944936: KO-DLN; Mus musculus; RNA-Seq
Mice were immunized with MOG/CFA to induce EAE. CD4+ YFP+ cells from spleen at the peak of disease were sorted on an AriaIII cell sorter into 15-ml tubes containing complete media, counted, and placed on ice. All samples were spun down at 500g for 10 min. The supernatant was removed, and cells were resuspended in 100 μl of 1 × PBS (Thermo Fisher Scientific) plus 0.04% BSA (Amresco). The cells were counted and checked for viability using a Luna dual fluorescence cell counter (Logos Biosystems). Cell counts ranged from 2× 105 to 4× 105 cells per millilitre and viability was greater than 98%. Libraries were prepared using the Chromium single cell 3′ v2 library and gel bead kit (10x Genomics). The complementary DNA content of each sample after cDNA amplification of 12 cycles was quantified and quality checked using a high-sensitivity DNA chip with a 2100 Bioanalyzer (Agilent Technologies) to determine the number of PCR amplification cycles to yield sufficient library for sequencing. After library quantification and quality checking by DNA 1000 chip (Agilent Technologies), during the library construction ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.