title: GSM7311081: DG, MeOH-fixed, rep1; Mus musculus; RNA-Seq
library_construction_protocol: DGs were dissected in PBS with 10mM glucose and individually subjected to an enzymatic and mechanical dissociation according to manufacturer's protocols (Neural Tissue Dissociation Kit P, Miltenyi Biotec). Single cells were obtained in DPBS wo Ca2+/Mg2+ containing 0.1 M D-(+)-Trehalose dihydrate at 4°C. The cell suspensions were pooled at equal ratios, and either MeOH-fixed or immediately processed for FAC-sorting. MeOH fixation was adapted from a protocol provided by 10X Genomics (CG000136). After dissociation, cells were transferred to 1.5 mL LoBind DNA Eppendorf tubes and pelleted at 300 rcf at 4°C for 5 min. The supernatant was removed carefully and the cell pellet resuspended in one volume of ice-cold DPBS wo Ca2+/Mg2+ containing 0.5 M D-(+)-Trehalose dihydrate, and four volumes of 100% MeOH pre-chilled to -20°C were added dropwise while gently mixing the cell suspension (final concentration:  106 cells/ml in 20:80 PBS/MeOH 0,1M Trehalose). The MeOH-fixed cells were kept at -20°C for 30 min and then stored at -80°C for 3 weeks. For rehydration, suspensions were moved from -80°C to 4°C on ice and kept for 5 min to equilibrate the temperature. Cells were pelleted at 1000 rcf at 4°C for 5 min, and resuspended in 3X SSC Buffer with 0.04% BSA, 0.2 U/µl of RNase inhibitor and 1 mM DL-Dithiothreitol. Subsequently, MeOH-fixed cells were processed either for FAC-sorting. After rehydration of the pooled MeOH-fixed cells (n = 4 mice), the cell suspension was centrifuged at 300 rcf at 4°C for 5 min and cells were resuspended in ice-cold DPBS wo Ca2+/Mg2+ containing 5% HEPES PUFFERAN® ≥99,5 %, p.a.. Next, single cells were FAC-sorted into 384-well microplates containing well-specific barcoded primers, ERCC spike-ins and reverse transcription reagents using a FACSAria™ Fusion cell sorter (BD Biosciences). Wells O21-O24 and P21-P24 were left empty as a no-template control. Different forward and side-scatter parameters were used for gating to discriminate single cells from debris and doublets or aggregates. To have the lowest bias between samples, control cell suspensions from age-matched mice (n = 3 mice) were freshly prepared, pooled and FAC-sorted the same day into another 384-well cell capture microplate. In this case, Sytox AADvanced™ Dead Cell Stain was added (5 min, at 4°C, in the dark) prior to FAC-sorting for excluding dead cells. The filled microplates were sealed and centrifuged at 2000 rcf for 2 min at 4°C, snap-frozen on dry ice and stored at -80°C. Libraries of microplates containing FAC-sorted cells from freshly-isolated and MeOH-fixed samples were prepared according to the CEL-Seq2 protocol. After cell lysis at 65°C, the RNA was reverse transcribed into cDNA followed by second-strand cDNA synthesis. The barcoded cDNA of all wells from one plate was pooled and amplified by In Vitro Transcription. The amplified RNA was fragmented, reverse transcribed into cDNA and again amplified using TruSeq small RNA primers (Illumina) to generate the final sequencing libraries. The quality and yield of the amplified RNA and the final cDNA libraries were checked on an Agilent Bioanalyzer. Thereafter, the libraries were paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1×75 bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles) aiming at 75000 reads/cell. Cel-Seq2