Nuclei were isolated from four E18 mouse combined cortex, hippocampus and ventricular zone tissues (C57BL/6 strain), obtained by 10x Genomics from BrainBits LLC. The E18 tissue was dissociated following Dissociation of Mouse Embryonic Neural Tissue for Single Cell RNA Sequencing (CG00055), then Nuclei Isolation from Cell Suspensions & Tissues for Single Cell RNA Sequencing (CG000124), and lastly Cell Multiplexing Oligo Labeling for Single Cell RNA Sequencing Protocols (CG000391).
Four E18 mouse nuclei samples were multiplexed at equal proportions with 3 CMOs per nuclei sample, resulting in a pooled sample labeled with 12 CMOs. Whole transcriptome/gene expression libraries were prepared following the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing User Guide (CG000388). The targeted gene expression libraries were generated as described in the Targeted Gene Expression Reagent Kits User Guide (CG000293) using a fully-custom mouse Neuroscience panel of ~110 genes.
Targeted sequencing data was processed using Cell Ranger, with web_summary.html and output files available in relevant tabs. The config.csv input file was submitted with four sample IDs (correlating to the four E18 mouse nuclei samples) and with three respective CMO IDs assigned per sample ID.
- Sequencing instrument: Illumina NovaSeq 6000
- Sequencing depth: 4,357 mean reads per cell
- Sequencing configuration: Paired-end (28 X 90), single-indexed. Read 1: 28 cycles (16 bp barcode, 10 bp UMI); i7 index: 10 cycles; i5 index: 10 cycles; Read 2: 90 cycles (transcript).
Pooled Multiplexed Sample - Key Metrics:
- Estimated number of cells: 24,151
- Cells assigned to a sample: 17,858