GSM8769124: twitcher, myeloid, P10, sample3; Mus musculus; RNA-Seq
Dissected hind-brains (brainstem and cerebellum) 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 into Trizol-LS using a BD FACS ARIA cell sorter equipped with a 100 um nozzle. Bulk RNA seq Libraries were generated using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (#E6420S), with 16-19 cycles of PCR cDNA amplification, which were experimentally determined using a dilution curve. Libraries were quality checked and quantified using an Agilent TapeStation using High Sensitivity D5000 ScreenTape (5067-5592), and High Sensitivity D5000 Reagents (5067-5593). Libraries were sequenced by NovaSeq6000 on an SP flowcell (Illumina), using sequencing chemistry v 1.5. We obtained at least 29x10^6 paired reads per sample, with a range of 29-42x106. A mean of 70.24% of reads were mapped across all samples.