Illumina NovaSeq 6000 paired end sequencing; Single cell analysis of CD45-positive immune cells infiltrating the central nervous system of mice undergoing experimental autoimmune encephalomyelitis.
C56BL/6 mice (10 to 12-week-old) were immunized subcutaneously at the base of the tail  with 100 g of MOG (3555) emulsified in complete Freund's adjuvant  containing 500 g of Mycobacterium tuberculosis. Pertussis toxin (200 ng; List Biological Laboratories) was injected intravenously at days 0 and 2 post-immunization. At day 17 post immunization, central nervous system CD45-positive immune cells were isolated and loaded onto a single channel of a 10x Chromium chip for each sample. The scRNA-seq libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10X Genomics) according to the manufacturers protocol. Gene expression (mRNA) libraries were constructed. Briefly, cells were counted and up to 20 000 cells were loaded in the 10X Genomics Chromium Controller to generate single-cell gel-beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq S2 flowcell (Illumina) with 26 cycles of read 1, 8 cycles of i7 index and 91 cycles of read 2, targeting a median depth of 50000 reads per cell for gene expression.