Nov 28, 2024
Nuclei Isolation and Clean-Up using GentleMACS and Anti-Nucleus Microbeads
- Fabian Köchl1
- 1Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland.
Protocol Citation: Fabian Köchl 2024. Nuclei Isolation and Clean-Up using GentleMACS and Anti-Nucleus Microbeads. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4w2k2vo5/v1
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: October 30, 2024
Last Modified: November 28, 2024
Protocol Integer ID: 111241
Abstract
Obtaining a clean nuclei suspension for subsequent single-nuclei RNA sequencing can be challenging. Especially liver nuclei suspensions are prone to high ambient RNA content.
Alternative nuclei clean up methods like Fluorescence-activated Cell Sorting can stress nuclei, decrease nuclei quality and are time consuming and need specialized equipment.
To overcome these challenges we applied a simple protocol for isolating and cleaning up nuclei for single-nuclei RNA sequencing using the GentleMACS Octo dissociator coupled with anti-Nucleus microbeads cleanup.
The following protocol has been successfully tested for Rat, Mouse and Minipig liver as well as Mouse brain.
Materials
gentleMACS‱ Octo Dissociator with Heaters (Miltenyi, # 130-096-427)
gentleMACS‱ Octo Coolers (Miltenyi,#130-130-533)
QuadroMACS Separator (Miltenyi,#130-090-976)
MACS MultiStand (Miltenyi, # 130-042-303)
Refrigerated Centrifuge
Anti-Nucleus MicroBeads (Miltenyi,# 130-132-997)
Nuclei Extraction Buffer (Mliltenyi# 130-128-024)
MACS BSA Stock Solution (# 130-091-376) or BSa 30% to be checked
MACS SmartStrainers (70 µm) (# 130-098-462)
MACS SmartStrainers (30 µm) (# 130-098-458) or CellTrics (Sysmex, #04-004-2326)
gentleMACS C Tubes (# 130-093-237, # 130-096-334)
LS Columns (# 130-042-401)
RNase inhibitor (e.g. RiboLock RNase Inhibitor )
Phosphate-buffered saline (PBS), pH 7.2
1. Preparations
1. Preparations
Pre-cool the buffers, and consumables with sample contact (e.g. gentleMACS C Tube and SmartStrainers) at 4 °C Overnight Place the gentleMACS Octo Coolers in -20 °C Overnight
Pre-cool the centrifuge to 4 °C before the start of the experiments
Per extraction, add RNase inhibitor (final concentration0.2 U/μL ) to pre-cooled 4 mL Nuclei Extraction Buffer.
Prepare Nuclei separation buffer (for resuspension) before further enrichment of the nuclei using Anti-Nucleus MicroBeads.
Per sample:
1x PBS: 10 ml
Nuclei Extraction Buffer: 1667 μL
BSA (30%): 15.6 μL
RNAse inhibitor (0.2U/ul): 58.3 μL
Note
Degas buffer before use, as air bubbles could block the column.
In a pre-cooled Petri dish on dry ice, cut a fresh frozen tissue sample into a piece of 25-50 mg with a pre-cooled scalpel.
Note
Make sure that the sample is always on dry ice.
Nuclei Extraction
Nuclei Extraction
Add 2 mL ice-cold lysis buffer to each pre-cooled gentleMACS C Tube.
Transfer tissue pieces to the gentleMACS C Tube containing lysis buffer
Close gentleMACS C Tube and place it on the gentleMACS Dissociator.
Retrieve gentleMACS Octo Coolers form the freezer and attach them over the C
tubes on the GentleMACs
Run gentleMACS Program 4C_nuclei_1 on the gentleMACS Dissociator. Detach the
tube after the program finished and place it on ice
Apply nuclei suspension to a MACS SmartStrainer (70 µm) (from brain tissue samples : 100uM) placed on
a 5 mL tube
Wash MACS SmartStrainer with 2 mL ice-cold lysis buffer
Discard MACS SmartStrainer and centrifuge nuclei suspension at 30 x g, 4°C, 00:05:00 .
Carefully aspirate supernatant completely.
5m
Resuspend nuclei pellet with 1.5 ml ice-cold resuspension buffer by slowly and gently
pipetting the sample up and down for 10 times
Apply nuclei suspension to a Celltrics Filter (30 μm)on a 1.5 mL tube or a MACS SmartStrainer (30μm) placed on a 5 mL tube.
Determine the nucleus number preferably using an automated Cell Counter
3 Magnetic Labelling
3 Magnetic Labelling
5m
5m
Transfer the volume that corresponds to 2 million nuclei to a new 1.5ml Eppi and
centrifuge300 x g, 4°C, 00:05:00 . Carefully remove supernatant
5m
Resuspend nucleus pellet in 900 µL of nuclei separation buffer and transfer to 5ml
tube
Add 100 µL of Anti-Nucleus MicroBeads
Mix well and incubate for 15 min 4 °C
Add 2 mL nuclei separation buffer and proceed to magnetic separation
Magnetic separation
Magnetic separation
Place LS column in the magnetic field of a suitable MACS Separator
Prepare column by rinsing with 3 mL of nuclei separation buffer
Apply nucleus suspension onto the column. Collect flowthrough containing debris
Wash column two times with 1 mL of nuclei separation buffer. Collect debris that
passes through and combine with the flow-through from previous step
Remove column from the separator and place it on a 1.5ml Eppi
Pipette 1 mL nuclei separation buffer onto the column. Immediately flush out the
magnetically labeled nuclei by firmly pushing the plunger into the column.
QC and preparation for Single Nucleus RNA sequencing
QC and preparation for Single Nucleus RNA sequencing
5m
5m
Centrifuge 300 x g, 4°C, 00:05:00 , remove supernatant
5m
Resuspend the pellet in 500 µL of PBS + 0.04% BSA (with RNAse inhibitor at 0.2U/ul).
Determine the nuclei concentration preferably using an Fluorescence-based automated Cell Counter
Dilute the sample with PBS + 0.04% BSA to the recommended concentration for
single nuclei RNA sequencing