title: GSM8371586: NLRP3 ko_3; Mus musculus; RNA-Seq
library_construction_protocol: Wild-type, NLRP3-/-, APP/PS1 and APP/PS1.NLRP3-/- mice were anaesthetized, sacrified and transcardially perfused with PBS.  The brains were removed and the hemispheres separated. One hemisphere was dissociated using the Neural Tissue Dissociation kit (P) (Miltenyi Biotec), and using MACS isolation the myelin was labelled and removed magnetically before the enrichment of CD11b+ microglia following the manufacturer's guidelines (Miltenyi Biotec). The cells were then stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Invitrogen) and CD11b (Invitrogen), and the CD11b+ live single cells were FACS sorted at 4°C (BD FACS Aria III) into 384-well plates containing 384 primers and Mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at -80°C. Viable single cells were FACS sorted into 384-well plates, called cell capture plates, that were ordered from Single Cell Discoveries, a single-cell sequencing service provider based in the Netherlands. Each well of a cell capture plate contains a small 50 nl droplet of barcoded primers and 10 µl of mineral oil (Sigma M8410). After sorting, plates were immediately spun and placed on dry ice. Plates were stored at -80° C.  Plates were shipped on dry ice to Single Cell Discoveries, where single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al. 2016 with primers described in van den Brink et al. 2017). Cells were heat-lysed at 65° C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol.3   to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles)