GSM7729200: DG, MeOH-fixed; Mus musculus; RNA-Seq
DGs of 5 mice were dissected as whole mounts in HBSS with calcium, magnesium and 10 mM glucose. Each DG was individually subjected to enzymatic and mechanical dissociation according to manufacturer's protocols (Papain Dissociation System, Worthington kit). Single cells were obtained in HBSS with calcium, magnesium and 0.1 M D-(+)-Trehalose dihydrate at 4 °C and kept on ice. After evaluation of the density and viability of the cell suspensions they were pooled at equal ratios and 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. P28 mouse MeOH-fixed cells were shipped on dry ice to Single Cell Discoveries. Prior to loading the 10x Chromium chip, cells were rehydrated as described in section 2.3, filtered and counted. Cells were loaded in a concentration of 700-1200 cells/µl on a 10x Genomics chip aiming for 10000 cells, and the chip was run on a Chromium controller to initiate GEM formation. Library preparation was performed following standard 10x Genomics guidelines 3' v3.1 workflow with feature barcoding (CG000317 guide) to generate gene expression libraries. The resulting cDNA libraries were paired-end sequenced on Illumina NovaSeq S4 Flow Cell chip on a NextSeq6000 platform (read 1: 28 cycles, index reads: 2×10 cycles, read 2: 90 cycles) to obtain an average of 70000 reads per cell   . FASTQ files were generated from BCL files with 10x Genomics Cell Ranger (Zheng et al., 2017), mkfastq 6.0. Read 1 was used for identification of cell barcode and UMI. Read 2 was used to map to the reference genome GRCm38 mouse. Filtering of empty barcodes was done following standard Cell Ranger procedures.  10x Genomics Kit 3' v3.1 (CG000317 guide)