3. Mix 2 mL Galbraith Buffer with 20 μL DTT, 20 μL RNasin,
10 μL RNAse inhibitor in the petri dish, and chop the sample
gently and quickly. Adjust sample amounts, chopping times,
and vary the volume of Galbraith Buffer to optimize the quantity and quality of nuclei. Filter the plant homogenate twice
through 30 μm Sysmex filter to avoid flow tip blockage.
4. Sort the nuclei immediately after adding PI (final concentration, 10 μg/mL), shortening the sorting time by increasing the
sheath pressure for higher sorting speed, with the aim of finishing sorting within 10 min. Generally, sorting 30,000 nuclei
could be done within 3–5 min.
5. Using a LoRetention tip, deposit 2 μL of the sorted nuclei
suspension onto the microscope slide and, without a coverslip,
observe the nuclei shape quickly using the phase contrast
microscope. Do this step quickly to avoid evaporation. The
quality of the sorted nuclei can be evaluated in terms of the
integrity of the nuclear membrane and percentage of the nuclei
with an intact membrane.
6. Measure the nuclear concentration using a LUNA-FL™ dual
fluorescence cell counter (similar products are acceptable) by
following the manual. Generally, !450 nuclei/μL can be
obtained.
7. Calculate the needed volume of sorted nuclei based on the
targeted nuclear number by considering the recovery rate,
transfer the nuclei suspension with a LoRetention tip to construct the libraries with Chromium Single Cell 3
0 Reagent Kits
v3 and a 10Â Genomics Chromium instrument according to
the manufacturer’s instructions. For 10,000 targeted nuclei,
44.5 μL containing 20,000 nuclei (450 nuclei/μL) are
employed.
8. Further processing of the libraries is done according to the
manufacturer’s instructions, and sequencing according to the
service that is employed. Gene-barcode matrices, obtained
using the 10ÂGenomics CellRanger pipeline [133], can be
processed using Seurat (https://www.cell.com/cell/fulltext/
S0092-8674(19)30559-8) or Monocle (https://www.nature.
com/articles/nbt.2859) for clustering of nuclei corresponding
to different cell types.
4 Notes
1. The measured nuclear CV values should always be close to the
lowest reported values, which for the CytoFlex are 1.32–1.77%
(Fig. 2). Higher values imply nuclear degradation of some type.
286
David W. Galbraith and Guiling Sun
10 μL RNAse inhibitor in the petri dish, and chop the sample
gently and quickly. Adjust sample amounts, chopping times,
and vary the volume of Galbraith Buffer to optimize the quantity and quality of nuclei. Filter the plant homogenate twice
through 30 μm Sysmex filter to avoid flow tip blockage.
4. Sort the nuclei immediately after adding PI (final concentration, 10 μg/mL), shortening the sorting time by increasing the
sheath pressure for higher sorting speed, with the aim of finishing sorting within 10 min. Generally, sorting 30,000 nuclei
could be done within 3–5 min.
5. Using a LoRetention tip, deposit 2 μL of the sorted nuclei
suspension onto the microscope slide and, without a coverslip,
observe the nuclei shape quickly using the phase contrast
microscope. Do this step quickly to avoid evaporation. The
quality of the sorted nuclei can be evaluated in terms of the
integrity of the nuclear membrane and percentage of the nuclei
with an intact membrane.
6. Measure the nuclear concentration using a LUNA-FL™ dual
fluorescence cell counter (similar products are acceptable) by
following the manual. Generally, !450 nuclei/μL can be
obtained.
7. Calculate the needed volume of sorted nuclei based on the
targeted nuclear number by considering the recovery rate,
transfer the nuclei suspension with a LoRetention tip to construct the libraries with Chromium Single Cell 3
0 Reagent Kits
v3 and a 10Â Genomics Chromium instrument according to
the manufacturer’s instructions. For 10,000 targeted nuclei,
44.5 μL containing 20,000 nuclei (450 nuclei/μL) are
employed.
8. Further processing of the libraries is done according to the
manufacturer’s instructions, and sequencing according to the
service that is employed. Gene-barcode matrices, obtained
using the 10ÂGenomics CellRanger pipeline [133], can be
processed using Seurat (https://www.cell.com/cell/fulltext/
S0092-8674(19)30559-8) or Monocle (https://www.nature.
com/articles/nbt.2859) for clustering of nuclei corresponding
to different cell types.
4 Notes
1. The measured nuclear CV values should always be close to the
lowest reported values, which for the CytoFlex are 1.32–1.77%
(Fig. 2). Higher values imply nuclear degradation of some type.
286
David W. Galbraith and Guiling Sun