TITLE: FIN-Seq (Frozen Immunolabeled Nuclei Sequencing)
AUTHORS: Ryoji Amamoto, Emanuela Zuccaro, Paola Arlotta, Constance L. Cepko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Thousands of frozen, archived tissues from postmortem human central nervous system (CNS) are currently available in brain banks. As single cell and single nucleus technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that transcriptional analysis of the human CNS requires cell type specificity. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples. Here, we developed FIN-Seq (</span><span style = ":UNDERLINE;">F</span><span>rozen </span><span style = ":UNDERLINE;">I</span><span>mmunolabeled </span><span style = ":UNDERLINE;">N</span><span>uclei </span><span style = ":UNDERLINE;">Seq</span><span>uencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types. </span></div></div>

[STEPS]
?. [Nuclei Extraction: ~1 hour]
Prepare all solutions and keep on ice with the Dounce homogenizer.
?. [Nuclei Extraction: ~1 hour]
Fill the glass Dounce homogenizer with 1 mL of cold homogenization buffer.
?. [Nuclei Extraction: ~1 hour]
Mince the tissue into little pieces and place in 1% PFA for 5 minutes on ice.
?. [Nuclei Extraction: ~1 hour]
Transfer the tissue pieces into the homogenizer. With the tight pestle, homogenize with 10-15 strokes on ice. Avoid foaming.
?. [Nuclei Extraction: ~1 hour]
Transfer the homogenate into a 5 mL polypropylene tube (Thermo Fisher 14-959-11A).
?. [Nuclei Extraction: ~1 hour]
Carefully add 2 mL of Sucrose Bed solution to the bottom of the tube so that the homogenate is above the sucrose solution.
?. [Nuclei Extraction: ~1 hour]
Spin at 500xg, 12 minutes, 4 degrees.
?. [Nuclei Extraction: ~1 hour]
Remove supernatant. Add 1 mL of 4% PFA solution and resuspend pellet. Incubate for 15 minutes at 4 degrees with rocking.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with Blocking Buffer. Incubate for 15 minutes at 4 degrees with rocking.
?. [Immunolabeling: ~2 hours]
Centrifuge at 2000xg for 5 minutes at 4 degrees. Remove supernatant.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with Primary Antibody in Blocking Buffer. Concentration depends on primary antibody. Generally, higher concentration than immunohistochemistry is necessary. Incubate for 30 minutes at 4 degrees with rocking.
?. [Immunolabeling: ~2 hours]
Centrifuge at 2000xg for 5 minutes at 4 degrees. Remove supernatant.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with Blocking Buffer. Incubate for 5 minutes on ice.
?. [Immunolabeling: ~2 hours]
Centrifuge at 2000xg for 5 minutes at 4 degrees. Remove supernatant.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with appropriate Secondary Antibody (1:1000) in Blocking Buffer. Incubate at 4 degrees for 30 minutes with rocking.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with Blocking Buffer. Incubate for 5 minutes on ice.
?. [Immunolabeling: ~2 hours]
Centrifuge at 2000xg for 5 minutes at 4 degrees. Remove supernatant.
?. [Immunolabeling: ~2 hours]
Resuspend pellet with Blocking Buffer.
?. [Nuclei Extraction: ~1 hour]
Centrifuge at 2000xg for 5 minutes at 4 degrees.
?. [FACS: Time depends on number of nuclei. ~30 minutes for 200,000 nuclei]
Gate based on Hoechst histogram, as shown below. This step will ensure that you get 2N nuclei. Make sure not to include 4N nuclei.
?. [FACS: Time depends on number of nuclei. ~30 minutes for 200,000 nuclei]
Gate using the plot with appropriate wavelengths. Often, the separation will not be as obvious as GFP or well characterized cell surface markers. Thus, it’s important to have proper controls.
?. [Immunolabeling: ~2 hours]
Filter and proceed to FACS.
?. [Representative FACS plots]
?. [Decrosslinking and RNA isolation: ~4 hours]
Spin at 3000xg for 7 minutes at 4 degrees.
?. [Decrosslinking and RNA isolation: ~4 hours]
Remove as much supernatant as possible.
?. [Decrosslinking and RNA isolation: ~4 hours]
From the Recoverall RNA/DNA Isolation Kit (Thermo Fisher Scientific AM1975), mix 100 uL of Digestion Buffer and 4 uL of protease for each sample. Adjust accordingly based on the volume of the leftover supernatant.
?. [Decrosslinking and RNA isolation: ~4 hours]
Incubate at 50 degrees for 3 hours (wrap the lid with parafilm). Note that this step differs from the manufacturer’s protocol.
?. [Decrosslinking and RNA isolation: ~4 hours]
The samples can be stored at -80 indefinitely after incubation or proceed to next steps according to the kit protocol.
?. [Decrosslinking and RNA isolation: ~4 hours]
Elute in ~17 uL of UltraPure water.
?. [Decrosslinking and RNA isolation: ~4 hours]
Store RNA at -80 degrees.
?. [FACS: Time depends on number of nuclei. ~30 minutes for 200,000 nuclei]
FACS isolated nuclei are sorted into Blocking Buffer and kept at 4 degrees.
?. [Decrosslinking and RNA isolation: ~4 hours]
It’s possible to run a RNA pico chip on the BioAnalyzer (Agilent), but RIN will not be accurate because rRNA is not enriched in nuclei. Qubit or BioAnalyzer can be used to estimate RNA concentration. If less than 10,000 nuclei, concentration may not be available by these methods.
?. [cDNA synthesis ]
Proceed to the SMART-Seq v.4 protocol for cDNA synthesis and amplification.
?. [cDNA synthesis ]
If RNA concentration is too low for Qubit or BioAnalyzer, >16 rounds of amplification may be necessary to generate enough cDNA. HS DNA chip should be run on the BioAnalyzer after the SMART-Seq v4 protocol and after Nextera indexing to ensure proper library construction.