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