GSM6407456: CMO_pool3; Mus musculus; RNA-Seq
Mice were perfused with cold PBS and cortical and hippocampal tissues were dissected and dissociated into a single cell suspension using the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677), according to the manufacturer's protocol.  Tissues were diced into 0.5 cm pieces and placed in the CTube contianing enzyme mix 1 followed by addition of enzyme mix 2.   Dissociation was performed by the gentleMACS Octo Dissociator using the 37C_ABDK_01 program.  Samples were filtered through a 70 µm strainer and sedimented at 300Xg for 10 minutes in 4C followed by centrifugation in debris removal solution at 3000Xg for 10 minutes in 4C.  After debris removal, cells were washed with cold D-PBS and centrifuged at 1000Xg for 10 mintues in 4C and resuspended in FACS buffer.  Four batches of single cell suspensions, with one replicate of each condition, were labeled with CD11b antibody and cell multiplex oligos per user guide (10X Genomics, CG000391 Rev A).  Equal proportions of cells from each sample were combined into one pool per batch and 100,000 live, CD11b+ cells were FACS sorted on FACS Aria III (BD Biosciences with a 100 μm nozzle) into 1% BSA in PBS. Single cell capture and library preparation was performed using Chromium Next GEM Single Cell 3ʹ Kit v3.1 per user guide (10X genomics, CG000388 Rev A).  Briefly, gel bead-in-emulsion (GEM) were generated by loading single cells in RT master mix onto the Chip G microfluidic along with partitioning oil and gel beads that contain an Illumina R1 primer sequence, Unique Moldecular Identifier, 10X barcode, and capture sequences for either mRNA transcripts or cell multiplex oligos.  After cDNA amplification of RT materials from the GEMs, transcriptional cDNA was separated from cell multiplex oligo cDNA via NucleoMag bead purification (Takara, 7444970.50), proceeded by library preparation of both sets of cDNA.  Transcriptional libraries were pooled equimolar for a sequencing run on an Illumina MiSeq instrument to estimate the number of cells captured in each library.  This MiSeq capture information was used to pool libraries in equal proportions for sequencing coverage of 50,000 reads per cell across libraries and 5000 reads per Cell multiplex oligo.  Sequencing on the Illumina NovaSeq 6000 instrument generating paired-end reads (28x10x10x90 bases) was performed by SeqMatic (Fremont, CA). 