Two approaches are described: the first involves sorting of
numerous protoplasts and nuclei, for this purpose being operationally defined as 50,000–100,000 per sample; we typically recover
100 ng total RNA from 100,000 arabidopsis nuclei. When sorting
these numbers, macroscopic amounts of sorted liquids (a mixture
of sheath and sample) accumulate with the sample. It is critical to
sufficiently dilute these liquids with chaotropic RNA-stabilizing
buffers to prevent transcript degradation. The second approach
involves sorting of individual protoplasts. In this case, the only
significant technical issue is ensuring that you can collect a single
sorted object, and quickly dilute it with the RNA-buffer.
3.5.1 Preparation of RNA
Targets from Populations of
Sorted Protoplasts and
Nuclei
Prepare RNA using the RNAqueous
®
-Micro Kit, which can accommodate an input sample volume of up to 0.1 mL. This procedure is
executed exactly as described by the manufacturer’s manual.
3.5.2 Preparation of RNA
Targets from Single Sorted
Protoplasts and Nuclei
This revised protocol is considerably simplified, in that it employs
commercial sequencing services that include single cell/nucleus
cDNA production and amplification steps. The single nuclei or
protoplasts are sorted into 10 μL of lysis buffer. The tube is flicked
to mix, centrifuged for 30 sec in a chilled microcentrifuge, sealed
and transferred onto dry ice. The tubes or plates are then shipped
for further processing at the location of the sequencing service. We
have found that the TaKaRa lysis buffer (catalog 635,013) containing 0.5 U/μL SUPERase In™ RNase Inhibitor (ThermoFisher
catalog number: AM2694) and 0.5 U/μL RNasin(R) RNase Inhibitor (Promega catalog number: N2111) works well for this purpose. Amplification and library preparation quality control steps are
then done according to the protocols of the specific sequencing
service.
3.6 Analysis of Gene
Expression in
Populations of Single
Nuclei using the
Chromium™ System
Sorting of populations of nuclei is enabled as previously described
in Subheadings 3.3.1 and 3.3.2. A total of 20,000 nuclei within a
total volume not exceeding 46.6 μL are required as the individual
input samples for Chromium processing, so it is recommended that
50–70 μm flow tips be employed for sorting, with sorting set to
single-droplet mode. Avoiding RNase contamination and processing the nuclei rapidly and on ice are important factors to obtain
high quality samples. Identifying appropriate chopping times, using
sharp razor blades, are also critical to obtain nuclei in high quantities and at high concentrations. Do not centrifuge the sorted
nuclei suspension to avoid adhesion.
1. Equip the sorter with a 75-μm (Beckman) or 70-μm (BD Aria)
nozzle, and get the sorter ready for nuclei sorting.
2. Rinse a 2-mL LoBind tube with 2% BSA and adjust its position
such that the droplets are deposited at the center of the bottom
of the tube, rather than on the tube wall.
Flow Cytometry and Sorting in Arabidopsis
285
numerous protoplasts and nuclei, for this purpose being operationally defined as 50,000–100,000 per sample; we typically recover
100 ng total RNA from 100,000 arabidopsis nuclei. When sorting
these numbers, macroscopic amounts of sorted liquids (a mixture
of sheath and sample) accumulate with the sample. It is critical to
sufficiently dilute these liquids with chaotropic RNA-stabilizing
buffers to prevent transcript degradation. The second approach
involves sorting of individual protoplasts. In this case, the only
significant technical issue is ensuring that you can collect a single
sorted object, and quickly dilute it with the RNA-buffer.
3.5.1 Preparation of RNA
Targets from Populations of
Sorted Protoplasts and
Nuclei
Prepare RNA using the RNAqueous
®
-Micro Kit, which can accommodate an input sample volume of up to 0.1 mL. This procedure is
executed exactly as described by the manufacturer’s manual.
3.5.2 Preparation of RNA
Targets from Single Sorted
Protoplasts and Nuclei
This revised protocol is considerably simplified, in that it employs
commercial sequencing services that include single cell/nucleus
cDNA production and amplification steps. The single nuclei or
protoplasts are sorted into 10 μL of lysis buffer. The tube is flicked
to mix, centrifuged for 30 sec in a chilled microcentrifuge, sealed
and transferred onto dry ice. The tubes or plates are then shipped
for further processing at the location of the sequencing service. We
have found that the TaKaRa lysis buffer (catalog 635,013) containing 0.5 U/μL SUPERase In™ RNase Inhibitor (ThermoFisher
catalog number: AM2694) and 0.5 U/μL RNasin(R) RNase Inhibitor (Promega catalog number: N2111) works well for this purpose. Amplification and library preparation quality control steps are
then done according to the protocols of the specific sequencing
service.
3.6 Analysis of Gene
Expression in
Populations of Single
Nuclei using the
Chromium™ System
Sorting of populations of nuclei is enabled as previously described
in Subheadings 3.3.1 and 3.3.2. A total of 20,000 nuclei within a
total volume not exceeding 46.6 μL are required as the individual
input samples for Chromium processing, so it is recommended that
50–70 μm flow tips be employed for sorting, with sorting set to
single-droplet mode. Avoiding RNase contamination and processing the nuclei rapidly and on ice are important factors to obtain
high quality samples. Identifying appropriate chopping times, using
sharp razor blades, are also critical to obtain nuclei in high quantities and at high concentrations. Do not centrifuge the sorted
nuclei suspension to avoid adhesion.
1. Equip the sorter with a 75-μm (Beckman) or 70-μm (BD Aria)
nozzle, and get the sorter ready for nuclei sorting.
2. Rinse a 2-mL LoBind tube with 2% BSA and adjust its position
such that the droplets are deposited at the center of the bottom
of the tube, rather than on the tube wall.
Flow Cytometry and Sorting in Arabidopsis
285