GSM8757516: microglia, WT, monocyte_transplanted, P35, sample1; Mus musculus; RNA-Seq
Dissected brains were placed in ice cold Medium A and homogenized with 4 strokes of a 7ml glass dounce homogenizer. The homogenate was filtered through a 70 μm filter. Homogenates were pelleted at 200 x g for 7 min. To remove myelin, the pellet was resuspended in Iso-percoll and spun 700 x g 20 min with no brake. The myelin layer was removed, and the remaining cell pellet was washed with Medium A. Cells were transferred to a u-bottom 96 well plate and blocked with CD16/CD32 (BD biosciences 553142) then stained for CD45 (103113, Biolegend) and CD11b (101228, Biolegend) and incubated with a dead cell detection dye (L34974, Invitrogen) on ice for 20 min. Following washes cells were sorted based on CD45, CD11b positivity (unmanipulated conditions) and GFP positivity (transplanted conditions) into PBS using a BD FACS ARIA cell sorter equipped with a 100 um nozzle. Cell viability after sorting exceeded 80%. Sorted cells were pelleted by centrifugation then resuspended in PBS to a final concentration of 1,000 cells per µl. Cells were loaded into a Chromium NextGem Chip G (10x Genomics) and inserted into a chromium controller to generate single-cell gel beads-in-emulsion (GEM). GEMs and scRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000121) according to the manufacturer's instructions. Briefly, GEM reverse-transcription incubation was performed. Next, GEMs were broken and complementary DNA (cDNA) was cleaned up with DynaBeads MyOne Silane Beads and SPRIselect beads. Barcoded cDNA was PCR amplified for twelve cycles. Following clean up with SPRIselect beads and enzymatic fragmentation, adaptor ligation, a single index primer was used to barcode samples (10x Genomics, PN-2000240 Single Index Plate T Set A). Sample index PCR was performed per the manufacturers cycling instructions for thirteen cycles. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using a 4150 TapeStation (Agilent). Sequencing libraries were loaded on an Illumina NovaSeq S2 flow cell with the following sequencing settings: read 1: 28 cycles; read 2: 90 cycles; index i7: ten cycles; index i5: ten cycles; 1.75 nM loading concentration.