Feb 19, 2022

Public workspaceIntranuclear CITE-seq (inCITE-seq): joint single-cell measurements of multiplexed nuclear proteins and RNA

This protocol is a draft, published without a DOI.
  • 1Broad Institute
Hattie Chung
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Protocol Citation: Hattie Chung, Emma Magee 2022. Intranuclear CITE-seq (inCITE-seq): joint single-cell measurements of multiplexed nuclear proteins and RNA . protocols.io https://protocols.io/view/intranuclear-cite-seq-incite-seq-joint-single-cell-bt7mnrk6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 13, 2021
Last Modified: February 19, 2022
Protocol Integer ID: 49101
Abstract
This protocol allows for intranuclear antibody staining of fixed nuclei in suspension. Nuclei suspensions are suitable for CITE-seq and 10X Genomic applications.
Materials
STOCK BUFFERS
  • All buffers should be fresh each time, except 0.2% PBST which can be made in advance, until ready for use.
  • Prepare all buffers prior to starting the protocol .
  • All buffer volumes below are for calculated for one sample + dead volume, except PBST which can be made in stock
  • Pre-chill and store all buffers TemperatureOn ice .


0.2% PBST
ABCD
Stock Conc.NameFinal Conc. Volume (mL)
10%Tween-200.2%10
-PBS-500
Total Volume (mL)510


EZ Lysis Buffer
ABCD
Stock Conc.NameFinal Conc.Volume (mL)
5,000URecombinant RNase Inhibitor (RRI)~1 U/µL0.011
EZ Lysis Buffer-11
Total Volume (mL)11.011


Resuspension Buffer (RSB)
ABCD
Stock Conc.NameFinal Conc.Volume (µL)
1MMgCl23mM3.3
5,000URecombinant RNase Inhibitor (RRI)~1 U/µL1.1
-PBS-1,095.6
Total Volume (µL)1,100.00


FA-NT Buffer
ABCD
Stock Conc.NameFinal Conc.Volume (µL)
16%Formaldehyde 1.33%257.69
10%Tween-200.1%31
10%NP-400.2%62
1MMgCl23mM9.3
-PBS-2,740.01


Blocking Buffer
ABCD
Stock Conc.NameFinal Conc.Volume (µL)
1,000UTruStain FcX™ PLUS Blocking Reagent1:10010
5%UltraPure BSA1%200
10%Dextran Sulfate (10% stock solution)0.05%5
1MMgCl23mM3
5,000URecombinant RNase Inhibitor (RRI)~1 U/µL1
-PBST-781
Total Volume (µL)1,000.00


Reagents used in this protocol:
  • ReagentDounce homogenizers SigmaCatalog #D8938-1SET
  • ReagentFalcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
  • ReagentFalcon® 5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Snap CapFalconCatalog # 352235
  • ReagentEppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204
  • ReagentpluriStrainer® 20 µmpluriSelectCatalog #43-50020-03
  • ReagentC-Chip disposable hemacytometerINCYTOCatalog #DHC-N01
  • ReagentRNase ZapSigma AldrichCatalog #R2020-250ML
  • ReagentNuclei EZ Lysis BufferSigmaCatalog #N-3408
  • ReagentRecombinant RNAse InhibitorTakarabioCatalog #2313A
  • Reagent1X PBS, cell culture gradeThermo Fisher Scientific
  • Reagent 1 M Magnesium Chloride (MgCl2)Sigma AldrichCatalog #M8266
  • Reagent10% Tween-20 SolutionTeknovaCatalog #T0710
  • ReagentNP-40 Surfact-Amps™ Detergent SolutionThermo FisherCatalog #28324
  • ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
  • ReagentTruStain FcX™ PLUS (anti-mouse CD16/32) AntibodyBioLegendCatalog #156603
  • ReagentUltraPure™ BSA (50 mg/mL)Thermo FisherCatalog #AM2618













Safety warnings
Formaldehyde and glacial acetic acid should be used in the chemical fume hood
Before you start the protocol
Before you start the protocol
20m
20m
All steps should be performed TemperatureOn ice or at Temperature4 °C . Cool a swinging bucket centrifuge to Temperature4 °C . Prepare all reagents and chill TemperatureOn ice .

Clean one set of douncers (mortar, pestle A and pestle B) for every sample as follows. Make sure to thoroughly wash inside the mortar and at the end of each pestle where the sample will come in contact:
  • Rinse with distilled water.
  • Spray with 70% ethanol. Let sit for ~Duration00:01:00
  • Rinse with distilled water
  • Spray with RNase Zap. Let sit for Duration00:01:00
  • Rinse with distilled water
  • Rinse with DNase and RNase free double distilled water
  • Let air dry on kimewipe

Once dry, pre-chill on TemperatureOn ice .

2m
Pre-chill all tubes TemperatureOn ice . For each sample, you will need:
  • 3 x 15mL Falcon Tubes
  • 1 x 1.5mL Eppendorf tube
  • 2 x 35µm filter-cap FACS tubes
  • 2 x 20µm filters

Nuclei Extraction
Nuclei Extraction
20m
20m
Remove frozen tissue sample from Temperature-80 °C storage and place on dry ice until ready.

Place sample into a clean, pre-chilled mortar filled with Amount2 mL of EZ Lysis Buffer.
Dounce with pestle "A" until resistance subsides (~40 strokes) TemperatureOn ice .

Place pestle "A" in 50mL Falcon tube to hold until ready for cleaning.
Douce with pestle "B" until resistance subsides (~40 strokes) TemperatureOn ice .

Place pestle "B" in 50mL Falcon tube to hold until ready for cleaning.
Transfer the Amount2 mL homogenate to a pre-chilled 15mL Falcon Tube.

Add Amount3 mL of EZ Lysis Buffer to raise the sample volume to Amount5 mL total -- volumes can be added to wash out the mortar before being added, to maximize nuclei transfer.

Incubate for Duration00:05:00 TemperatureOn ice .


5m
During this incubation, spray mortars and pestles with 10% bleach, let sit, and rinse with distilled water in order to clean off remaining tissue. After rinsing, soak mortars and pestles in 10% bleach and store until next use.
Spin down nuclei at Centrifigation500 x g, 4°C, 00:05:00 in pre-cooled swinging bucket centrifuge.

5m
Carefully remove and discard supernatent.
Resuspend pellet in Amount1 mL of EZ Lysis Buffer using a P1000 pipette. Mix carefully and thoroughly. Add another
Amount4 mL of EZ Lysis Buffer. Mix carefully and thoroughly. The total volume should be Amount5 mL .

Incubate for Duration00:05:00 TemperatureOn ice .
5m
Spin down nuclei at Centrifigation500 x g, 4°C, 00:05:00 in pre-cooled swinging bucket centrifuge.

5m
Carefully remove and discard supernatent.
Resuspend pellet in Amount1 mL of RSB Buffer.

Filter Amount1 mL of nuclei suspension through a pre-chilled Thikness35 µm filter cap FACS tubes.
Fixation and Permeabilization
Fixation and Permeabilization
Transfer Amount1 mL of nuclei suspension from the filter tube into a pre-chilled 15mL Falcon tube.

Add Amount1 mL of FA-NT solution using a P1000 pipette, mixing the first Amount1 mL carefully and thoroughly with the nuclei suspension. Add Amount2 mL more of FA-NT solution. The total volume in the tube should be Amount4 mL .

Immediately spike Amount3 µL of glacial acetic acid to nuclei suspension.

Incubate for Duration00:10:00 at Temperature4 °C while rocking.

10m
Immediately after, quench the fixation reaction by adding Amount3 µL of Concentration1 M Glycine. Use P1000 pipette to mix sample thoroughly to ensure equal distribution of glycine throughout solution.

Filter through a Thikness20 µm filter into new, pre-chilled 15mL Falcon Tube.

Spin down nuclei at Centrifigation850 x g, 4°C, 00:05:00 .

5m
Remove and discard supernatent.
Primary Antibody Stain
Primary Antibody Stain
Resuspend pellet in Amount500 µL of Blocking Buffer. Pipette up and down multiple times using a P200 pipette to ensure the sample is mixed thoroughly.

Incubate for Duration00:15:00 at Temperature4 °C while rocking.

15m
Spin down nuclei at Centrifigation850 x g, 4°C, 00:05:00 .

5m
Remove and discard supernatent.
Resuspend pellet in Amount200 µL of primary antibody diluted to the appropriate concentration in Blocking Buffer.
Incubate for Duration01:00:00 at Temperature4 °C while rocking.

1h
Spin down nuclei at Centrifigation850 x g, 4°C, 00:05:00 .
5m
Carefully remove and discard supernatent.
Resuspend pellet in Amount500 µL of PBST.

Incubate Duration00:05:00 TemperatureOn ice .

5m
Repeat wash steps 34-37 to to ensure removal of any excess antibodies.
10m
Spin down nuclei at Centrifigation850 x g, 4°C, 00:05:00 .

5m
Remove and discard supernatent. If preforming a secondary antibody stain, proceed to step 42. If loading onto 10x, proceed to step 50.
[OPTIONAL] Secondary Antibody Staining
[OPTIONAL] Secondary Antibody Staining
Resuspend pellet in Amount200 µL of secondary antibody diluted 1:1000 in Blocking Buffer.

Incubate for Duration00:20:00 at Temperature4 °C while rocking.
20m
After incubation, spike in Amount2 µL of 100x DAPI into each sample. Mix carefully and thoroughly using a P200 pipette.

Repeat wash steps 34-37 two times to ensure removal of any excess antibodies in solution.
10m
Spin down nuclei at Centrifigation850 x g, 4°C, 00:05:00 .
5m
Resuspend pellet in Amount500 µL of PBST.

filter through a 20Thikness20 µm filter into a pre-chilled FACS tube.

Keep samples in dark TemperatureOn ice until processing via flow cytometry.

preparation for 10x loading
preparation for 10x loading
Resuspend pellet in Amount100 µL of pre-chilled RSB Buffer (ensure that there is no Tween-20 in this solution!) using a P200 pipette. Mix carefully and thoroughly. Add Amount200 µL more of RSB using a P1000 pipette. Mix carefully and thoroughly. Total volume should be Amount300 µL .

Filter through a Thikness20 µm filter into a pre-chilled FACS tube.

Count nuclei using hemocytometer chamber. Keep nuclei TemperatureOn ice until ready for 10X loading.