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
This protocol describes isolation of nuclei from left and right 10 week old mouse kidneys (tissue ID: 09) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for 1. single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq) and 2. single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. For 8 samples, this protocol takes about 3.5 hours from start to finish.
The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/ul, and 1 fixed nuclei pellet pooled across all 8 strains for SHARE-seq, all stored at -80C.
The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. When nuclei are extracted and counted, we determine whether we have enough to fix for Split-seq and SHARE-seq and set aside 4 million and 1 million, respectively. Ideally, the second and third parts of this protocol are performed in parallel by at least two technicians to save time. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents (see attachment for original version). The third part describes nuclei fixation using a modified version of the SHARE-seq fixation protocol (see attachment for original version). Any remaining nuclei are flash-frozen as a dry pellet and stored at -80C.
We recommend using a 5 ml pipette for aspirations and resuspensions > 1 ml.
Record everything in the IGVF spreadsheet, “Samples into experiment” tab.
Materials
Name
Manufacturer
Cat #
Nuclei Fixation Kit v2
Parse Biosciences
ECF2003
Nuclei Extraction Buffer
Miltenyi Biotec
130-128-024
RNase Inhibitor, murine
New England Biolabs
M0314L
PBS
HyClone
SH30256.02
7.5% BSA
Life Technologies
15260037
1 M HEPES pH 7.3
Sigma
H0887-100ml
NaCl
Fisher
BP358-1
MgCl2
Fisher
AA12315A7
Tween-20
Fisher
BP337-500
5% digitonin
Promega
G944A
Enzymatics RI
Enzymatics
Y9240L
SUPERase RI
Invitrogen
AM2696
Yeast tRNA
Invitrogen
AM7119
Glycine
Fisher
BP381-500
1M Tris pH 8.0
Thermo
AM9855G
Formaldehyde (methanol-free)
EMS
15710
gentleMACS C Tube
Miltenyi Biotec
130-093-237
gentleMACS Octo Dissociator
Miltenyi Biotec
130-095-937
MACS SmartStrainers (70 um)
Miltenyi Biotec
130-110-916
MACS SmartStrainers (30 um)
Miltenyi Biotec
130-098-458
NucBlue Fixed Cell ReadyProbes
Thermo Fisher
R37606
Hemacytometer
Fisher Scientific
02-671-51B
Mr. Frosty
Sigma-Aldrich
635639
Reagents/equipment, manufacturer and catalog number
Name
reagent
Volume for 8 samples
Final concentration
Lysis buffer
Nuclei Extraction Buffer
40 ml
NA
40 U/ul RNase inhibitor
200 ul
0.2 U/ul
NB-BSA + RNase inhibitor (make 2 aliquots)
Nuclei Buffer (Parse Biosciences)
3.15 ml
NA
7.5% BSA
350 ul
0.75%
RNase inhibitor (Parse Biosciences)
44.1 ul
RSB
PBS
42 ml
7.5% BSA
560 ul
0.1%
RNase inhibitor
210 ul
0.2 U/ul
SHARE-RSB
1 M HEPES pH 7.3
150 ul
10 mM
5 M NaCl
30 ul
10 mM
1 M MgCl2
45 ul
3 mM
10% Tween-20
150 ul
0.1%
H2O
14.625 ml
7.5% BSA
80.26 ul
0.04%
5% digitonin
30 ul
0.01%
Enzymatics RI
37.5 ul
0.1 U/ul
SUPERase RI
18.75 ul
0.025 U/ul
Yeast tRNA
150 ul
100 ug/ml
Buffers
Setup
Setup
Coat SHARE-seq nuclei prep tubes with BSA. Fill 8 1.5 ml tubes with 1.5 ml 1% BSA in H2O and incubate for 30 minutes. After incubation, aspirate BSA solution and dry for 30 minutes. Store at 4C.
Label tubes.
Pre-chill centrifuge to 4C.
Prepare ice buckets.
Prepare 40 ml lysis buffer in a 50 ml conical tube on ice. Distribute 2.5 ml into 8 gentleMACS C Tubes on ice. Add 200 ul RNase inhibitor to the lysis buffer aliquot the day of the experiment.
Prepare 42 ml RSB in a 50 ml conical tube on ice. Add 210 ul RNase inhibitor the day of the experiment.
Prepare 2 aliquots of 3.5 ml NB + BSA. Add 44.1 ul RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment to each aliquot.
Prepare 2.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 31.5 ulRNase inhibitor to 2.5 ml nuclei buffer.
Prepare 15 ml SHARE-RSB in a 50 ml conical tube at room temperature. To SHARE-RSB, add 30 ul digitonin, 37.5 ul Enzymatics RI, 18.75 ul SUPERase RI, and 150 ul yeast tRNA fresh the day of the experiment.
Thaw components of 2Parse Biosciences Nuclei Fixationv2 kits at room temperature, then place on ice.
Distribute 20 ul NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei.
Tissue lysis and nuclei extraction
Tissue lysis and nuclei extraction
Keep flash frozen tissue samples on dry ice until lysis.
Drop whole frozen tissue into a chilled gentleMACS C Tube with 2.5 ml lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. There should be 2 kidneys.
Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice.
Filter nuclei suspension through 70 um MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering.
Wash 70 um MACS SmartStrainer with 2 ml additional lysis buffer. Add 2 ml to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer.
Discard strainer and centrifuge the 4.5 ml nuclei suspension at 4C, 350g for 5 minutes.
Discard supernatant and resuspend nuclei pellet in 3 ml RSB.
Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube.
Dilute some nuclei 1:10 by adding 200 ul nuclei to 1.8 ml RSB in a new 5 mL tube. This should help the concentration reach around 4 million per ml.
Count 1:10 diluted nuclei. Use 1:11 dilution factor, 2 ul + 20 ul dye. Final dilution factor = 1:110.
Parse nuclei fixation
Parse nuclei fixation
Set aside 4 million nuclei in RSB in a new 5 ml tube and spin down at 4C, 350g for 5 minutes.
Remove supernatant and and resuspend nuclei in 750 ul NB-BSA + RNase inhibitor and filter through a 40 um strainer (provided in Parse Biosciences kit) into a new 5 ml tube.
Add 250 uLNuclei Fixation Solution and mix 3 times. Do not over-mix.
Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 ul and keep the P1000 at 250 ul.
Add 80 uL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 uL. Do not over-mix.
Incubate 3 minutes with nuclei on ice.
Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix.
Centrifuge at 4C, 750g for 10 minutes.
Aspirate and discard supernatant.
Resuspend the samples in 300 ul Nuclei Buffer with RNase inhibitor without BSA and move through a 40 um filter into a labeled 1.5 ml tube.
Count nuclei. Use 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
Add Nuclei DMSO: 5 uL and gently flick tubes to mix. One minute later, add another 5 uL and flick to mix, then after another minute add a final 5 uL for a total volume of 15 uL. Mix by gently pipetting 5x with a P200 set to 125 ul.
Split nuclei suspension into 2 labeled tubes, 150 ul per tube.
Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks.
SHARE-seq nuclei fixation
SHARE-seq nuclei fixation
Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4C, 750g for 5 minutes.
Remove supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack.
At RT, add 13.34 ul of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate cells at RT for 5 minutes.
To quench fixation, per reaction, add 56.1 ul fresh 2.5M Glycine (0.94g per 5 ml stock), 50 ul of 1M Tris pH 8.0, 13.3ul of 7.5% BSA, and mix using a pipette. Incubate on ice for 10minutes.