GSM9078251: isolated Microglia-set 1, hashtaged for the different conditions; Mus musculus; RNA-Seq
Hippocampal tissue was chopped and mechanically dissociated in HBSS with 15 strokes using a Ddounce homogenizer usingwith a loose pestle. The supernatant was transferred to a falcon and the cell pellet was again pulsed 10-15 times with the tight pestle in HBSS. The supernatant was again collected and filtered through a 70µm strainer followed by 40% Percoll density gradient centrifugation at 500g for 30 min at 4˚C. Cell pellet was collected and blocked with CD16/32 for 30min and then stained with live/dead BD700 and the following antibodies: CD45-BV421, CD11b-PE-Cy7, Cd11c-FITC, F4/80 BV786, Celc7a-PE  and Ly6C APC-Cy7. Cells from the four different conditions were stained with 8 Totalseq-A anti-mouse hashtag antibodies and sorted with a FACS device to pool equal numbers of cells from each individual. All sorted cells (~8,000) were carefully mixed with reverse transcription mix before loading cells on the 10X Genomics Chromium controller in a Chromium Single Cell G Chip targeting ~4,800 cells per reaction. The droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (10X Genomics). After amplification of cDNA with 13 cycles using primers to enrich cDNA as well as Totalseq-A hashtag sequences, the samples underwent a purification with SPRIselect beads (Beckman Coulter) with 0.6x volume of beads to enrich cDNA fragments (>400 bp), followed by an additional purification of the supernatant with 2x volume of SPRI beads to separately enrich the small fragments from the hashtag sequences. After quality check and quantification of the cDNA on the Fragment Analyzer (using the DNF-473 NGS Fragment Kit, Agilent), the 10X Genomics single cell RNA-seq library preparation - involving fragmentation, dA-Tailing, adapter ligation and 14 cycles indexing PCR – was performed based on the manufacturer's protocol. In parallel, the hashtag library was prepared by a 12 cycles index PCR with the 10x Genomics TT-index set A. After quantification, both libraries were sequenced on Illumina NovaSeq 6000 S4 flowcells in 200 bp paired-end mode, generating 125 million fragment pairs for the gene expression libraries and 55 million fragment pairs for the hashtag library.