Journal: STAR Protocols

Article Title: Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing

doi: 10.1016/j.xpro.2022.101337

Figure Lengend Snippet:

Article Snippet: Transfer 2 μL (700 pg) of the amplified cDNA in an empty 384-well plate. c. Desiccate the liquid completely by vacuum centrifuge for 15 min at 30°C. d. In the meantime, thaw all reagents from the Tagmentation Master Mix A on ice and, once thawed, prepare the mix as described in the section. e. Add 1 μL of Tagmentation Master Mix A with a nanodispenser to each well, heat-seal the plate for 1 s at 166°C, vortex vigorously and spin it down. f. Incubate the plate in a pre-heated thermal cycler using the following program. g. To amplify the tagmented DNA, add 3 μL of the KAPA High-Fidelity Hot Start ReadyMix (2×) and 2 μL of P5 and P7 barcoded Nextera amplification primers (1 μM of each) to a total reaction volume of 6 μL.

Techniques: Recombinant, Sterility, Red Blood Cell Lysis, Magnetic Beads, Reverse Transcription, Software, Ointment, Adhesive

Journal: STAR Protocols

Article Title: Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing

doi: 10.1016/j.xpro.2022.101337

Figure Lengend Snippet: Tagmentation Master mix A (Vazyme TruePrepTM DNA Library Prep Kit V2 for Illumina)

Article Snippet: Transfer 2 μL (700 pg) of the amplified cDNA in an empty 384-well plate. c. Desiccate the liquid completely by vacuum centrifuge for 15 min at 30°C. d. In the meantime, thaw all reagents from the Tagmentation Master Mix A on ice and, once thawed, prepare the mix as described in the section. e. Add 1 μL of Tagmentation Master Mix A with a nanodispenser to each well, heat-seal the plate for 1 s at 166°C, vortex vigorously and spin it down. f. Incubate the plate in a pre-heated thermal cycler using the following program. g. To amplify the tagmented DNA, add 3 μL of the KAPA High-Fidelity Hot Start ReadyMix (2×) and 2 μL of P5 and P7 barcoded Nextera amplification primers (1 μM of each) to a total reaction volume of 6 μL.

Techniques: Concentration Assay

Journal: STAR Protocols

Article Title: Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing

doi: 10.1016/j.xpro.2022.101337

Figure Lengend Snippet: Tagmentation Master Mix B (Illumina DNA prep BLT Tagmentation)

Article Snippet: Transfer 2 μL (700 pg) of the amplified cDNA in an empty 384-well plate. c. Desiccate the liquid completely by vacuum centrifuge for 15 min at 30°C. d. In the meantime, thaw all reagents from the Tagmentation Master Mix A on ice and, once thawed, prepare the mix as described in the section. e. Add 1 μL of Tagmentation Master Mix A with a nanodispenser to each well, heat-seal the plate for 1 s at 166°C, vortex vigorously and spin it down. f. Incubate the plate in a pre-heated thermal cycler using the following program. g. To amplify the tagmented DNA, add 3 μL of the KAPA High-Fidelity Hot Start ReadyMix (2×) and 2 μL of P5 and P7 barcoded Nextera amplification primers (1 μM of each) to a total reaction volume of 6 μL.

Techniques: Concentration Assay

Journal: Nature neuroscience

Article Title: Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

doi: 10.1038/nn.4216

Figure Lengend Snippet: ( a ) Experimental workflow starts with the isolation, sectioning and micro-dissection of the primary visual cortex from a transgenic mouse. The tissue samples are converted into a single-cell suspension, single cells are isolated by FACS, poly(A)-RNA from each cell is reverse transcribed (RT), cDNA is amplified and fragmented, and sequenced on a next-generation sequencing (NGS) platform. ( b ) Analysis workflow starts with the definition of high variance genes and iterative clustering based on two different methods, PCA (shown here) and WGCNA, and cluster membership validation using a random forest classifier. Cells that are classified consistently into one cluster are called ‘core’ cells (N = 1424), while cells that are mapped to more than one cluster are labeled ‘intermediate cells’ (N = 255). After the termination criteria are met, clusters from the two methods are intersected, and iteratively validated until all core clusters contain at least 4 cells. ( , ). ( c ) The final 49 clusters were assigned an identity based on cell location and marker genes . Each type is represented by a color bar with the name and number of core cells representing that type. The violin plots represent distribution of mRNA expression on a linear scale, adjusted for each gene (max. RPKM on the right), for major known marker genes: Snap25 (pan-neuronal); Gad1 (pan-GABAergic); Vip, Sst and Pvalb (GABAergic); Slc17a7 (pan-glutamatergic); Rorb (mostly L4 and L5a); Foxp2 (L6); Aqp4 (astrocytes); Pdgfra (oligodendrocyte precursor cells, OPCs); Mog (oligodendrocytes); Itgam (microglia); Flt1 (endothelial cells) and Bgn (smooth muscle cells, SMC).

Article Snippet: We developed a robust procedure for isolating individual adult live cells from the suspension by fluorescence activated cell sorting (FACS), reverse transcribed and amplified full-length poly(A)-RNA with the SMARTer protocol, converted the cDNA into sequencing libraries by tagmentation (Nextera XT), and sequenced them by next generation sequencing ( , , ).

Techniques: Isolation, Dissection, Transgenic Assay, Suspension, Reverse Transcription, Amplification, Next-Generation Sequencing, Biomarker Discovery, Labeling, Marker, Expressing