scRNA-Seq of mus musculus: 2wks Female
The single cell gene expression analysis is conducted using a 10X Chromium single cell system (10X Genomics, Inc) and a NovaSeq 6000 sequencer (Illumina, Inc). Depending on the quality of the initial cell suspension, the single cell preparation may include re-suspension, centrifugation, and filtration to remove cell debris, dead cells and cell aggregates. The final single cell suspension should be washed 3 times with either PBS buffer or preferred media. Each clean single cell suspension will then be counted with hemocytometer under microscope for cell number and cell viability. The 10 single cell suspensions have a viability of 68-91% and minimal cell debris and aggregation. Ten thousand targeted cell recovery per sample is applied to a single cell master mix with lysis buffer and reverse transcription reagents, following the Chromium Single Cell 3 Reagent Kits V3 User Guide, CG000183 Rev A (10X Genomics, Inc). Along with the single cell gel beads and partitioning oil, the single cell master mixture containing the single cell suspension is dispensed onto a Single Cell Chip B in separate wells, and the chip loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library will be examined by Bioanalyzer and Qubit. The resulting library will be sequenced in a custom program for 28b plus 91b paired-end sequencing on Illumina NovaSeq 6000. Depending on targeted cell recovery, about 50K reads per cell will be generated and 91% of the sequencing reads reached Q30 (99.9% base call accuracy). A Phred quality score (Q score) will be used to measure the quality of sequencing.