Single nuclei RNA sequencing

Overview

This protocol is designed to isolate nuclei from frozen ovarian cancer tumor specimens and sequence the nuclei using the 10X Genomics platform. The protocol is based on multiple sources including Gaublomme, et al., Nature Communications 2019, Habib, et al., Nature Methods 2017, BioLegend TotalSeq protocols, 10X Nuclei Prep Protocol,

Materials and Reagents

• Dounce Homogenizers (Sigma, Cat # D8938-1SET) (Note, two pestles required per sample)
• Filters (30 µm) (Miltenyi Biotec 130-041-407)
• Filters (20 µm) (Miltenyi Biotec 130-101-812)
• Lo-bind microfuge 1.5 ml (Sigma-Aldrich Z666505-100EA)
• Protector RNase Inhibitor (Sigma-Aldrich 3335399001)
• DAPI (ThermoFisher Scientific D1306)
• Hashtag anti-nuclear pore antibodies (Biolegend, TotalSeq MAb414 antibodies)
• Fc Blocking reagent (Biolegend 422302)

Methods

Preparation of tumor tissue for snap freezing and storage at -80ºC

• Perform procedures in a sterile hood, keeping tissue at 4ºC at all times.
• Transport tumor specimen from pathology lab to Starr lab on ice in a 50 ml conical with DMEM.
• Weigh the bulk tumor specimen.
• Wash specimen with ice cold PBS in a petri dish sitting on ice. Remove excess PBS
• Use a sterile scalpel and/or scissors to remove fatty and necrotic tissue.
• Weigh the cleaned tumor specimen
• Cut the specimen into pieces required for all types of analyses. For the portion used for single nuclei sequencing, cut into pieces ~50-200 mg each.
• Place the tissue pieces in a 1.5 ml cryovial and snap freeze in liquid nitrogen
• Store cryovials at -80ºC or in liquid nitrogen.

Preparation of buffers

• Prepare fresh NSLB, STWB and STB buffers using the recipes below

Isolate nuclei

• For each sample to barcode and pool, prepare a separate dounce homogenizer with two pestles (A & B)
• Add 1 ml NSLB to the dounce homogenizers and place on ice
• Remove a 50-200 mg section of frozen tumor specimen from the cryovial
• Mince tissue on a petri dish sitting on ice and place minced pieces in the homogenizer with tweezers
• Dounce 20 times with pestle A and then repeat 20 times with pestle B
• Add 1 ml of STWB to homogenizer and decant through a 30 µm filter into a 15 ml conical
• Rinse homogenizer with 1 ml of STWB and decant through a 30 µm filter into the same 15 ml conical. Repeat two more times. At this point there should be ~5 ml of homogenate in the 15 ml conical.
• Centrifuge at 500g for 5 min at 4ºC to pellet the nuclei.
• Carefully remove the supernatant with a pipette
• Resuspend the nuclei pellet in 200 µl of STWB and transfer to a 1.5 ml lo-bind centrifuge tube by passing through a 20 µm filter.

Count nuclei

• Prepare a DAPI solution by dissolving 250 µg DAPI in 100 µl STB
• Mix 20 µl DAPI solution with 20 µl of the nuclei suspension
• Place 10 µl on hemocytometer slide and count nuclei in microscope
• Dilute or concentrate solution to 1-2e6 nuclei/100 µl STB buffer
• Optional: Use Trypan blue and count manually or using an automated cell counter

Stain Nuclei with anti-nuclear pore hashtag antibodies

• Add 10 µl Fc Blocking reagent to 100 µl of nuclei solution (1-2e6 nuclei/100 µl) in a lo-bind microfuge tube and incubate 5 min at 4ºC
• Add 1 µg of single nuclei hashing antibody to the tube and incubate for 10 min at 4ºC
• Wash nuclei 3x with 1.2 ml STB by centrifuging at 500g for 5 min at 4ºC.
• Make a final resuspension of 500 to 3,000 nuclei/µl in STB
• Filter nuclei through 20 µl filter. Count nuclei and adjust concentration to 500 to 3,000 nuclei/µl

Nuclei capture using 10X Genomics platform

• Use 14μl of pooled sample as input into the 10X Genomics single-cell 3’ v2 assay and process as described until before cDNA amplification
• To be determined: Follow specialized protocol for cDNA amplification (see supplementary methods in Gaublomme and BioLegends protocols

Notes

• When washing and resuspending nuclei, always use sufficient volumes to maintain concentrations of less than 5000 nuclei/μl (i.e. 5 million nuclei resuspended in 1 ml Nuclei Washing and Resuspension Buffer). Maintaining nuclei at higher concentrations may cause aggregation and clumping
• To maximize the likelihood of achieving the desired recovery target, the optimal input nuclei concentration for 10X is 700 – 1200 nuclei/μl.
• Can adjust BSA in the buffers from 0.01% to 2.0%
• Can adjust RNase inhibitor concentration