GSM5353267: WT 5xFAD-T-Ts-; Mus musculus; RNA-Seq
Processing and tissue-collection of whole brain was performed as previously described (Van Hove et al., 2019, doi: https://doi.org/10.1038/s41593-019-0393-4; Shemer et al., 2020, doi: https://doi.org/10.1016/j.immuni.2020.09.018, Kim et al., 2021, doi: https://doi.org/10.1016/j.immuni.2020.11.007). From these tissue samples, CD45+ FACS sorted immune cells were loaded on a Chromium Chip B (10x Genomics, No.1000074), or Chip G (10x Genomics, No. 2000177) on a GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEMs  were prepared using the GemCode Single Cell 3' Gel Bead according to the manufacturer's instructions. Briefly, GEM reverse-transcription incubation was performed in a 96-deep-well reaction module at 53°C for 45 min, 85°C for 5 min and ending at 4°C. Next, GEMs were broken and complementary DNA (cDNA) was cleaned up with DynaBeads MyOne Silane Beads (10x Genomics, No. 2000048) and SPRIselect Reagent Kit (Beckman Coulter, No. B23318). Full-length, barcoded cDNA was PCR amplified with a 96-deep-well reaction module at 98°C for 3 min, eleven cycles at 98°C for 15 s, 63°C for 20 s and 72°C for 1 min, followed by one cycle at 72°C for 1 min and ending at 4°C. Following cleaning up with the SPRIselect Reagent Kit and enzymatic fragmentation, library construction to generate Illumina-ready sequencing libraries was performed using the Library Kit (v3 and v3.1, 10xGenomics, No. 1000075) and the Chromium i7 Multiplex Kit (10x Genomics, No. 120262), according to the manufacturer's instructions. R1 (read 1 primer), P5, P7, i7 sample index and R2 (read 2 primer sequence) were added via end-repair, A-tailing, adapter ligation, post-ligation SPRIselect cleanup/size selection and sample index PCR. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent).