GSM9018244: main, Tet2-/- Tx 5xFAD, rep2; Mus musculus; RNA-Seq
Mice were perfused with 20mL cold PBS prior to brain extraction. Brains were minced in dissociation buffer and incubated for 45mins at 37˚C. Homogenized cells were then subjected to a Percoll gradient to remove myelin. Cells were then filtered and enriched for either CD11a or CD45 using magnetic beads. Library preparation was carried out using 10X Genomics manufacturers protocol CG000317_ChromiumNextGEMSingleCell3_v3.1_CellSurfaceProtein. Briefly, GEMs (Gel Beads-in-emulsion) were generated by combining barcoded Single Cell 3' v3.1 Gel Beads, a Master Mix with cell surface protein labeled cells, and Partitioning Oil onto Chromium Chip G. Immediately following GEM generation, the Gel Bead is dissolved releasing the three types of primers and any co-partitioned cell is lysed. The poly(dT) and one of the capture sequence primers in the gel bead are engaged simultaneously in two different reactions inside individual GEMs. Both are mixed with cell lysate and Master Mix containing RT reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA and barcoded DNA from the cell surface protein Feature Barcode. After incubation, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the cell barcoded products from the post GEM-RT reaction mixture. The cell barcoded cDNA molecules are amplified via PCR to generate sufficient mass for library constructions. Size selection is used to separate the amplified cDNA molecules for 3' Gene Expression and Cell Surface Protein library construction. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.