TITLE: BIDMC TMC - low input single nuclei sequencing isolation (NNLB)
AUTHORS: Luciano G Martelotto, aploumak, Shuoshuo Wang, Nikolaos Kalavros, Ioannis Vlachos

[DESCRIPTION]
Single nuclei isolation protocol used by the BIDMC TMC to isolate intact nuclei from very low input tissues. Has been used successfully by the BIDMC TMC to isolate and sequence nuclei from isolated peripheral lymphatic vessels. Developed in collaboration with Dr. Luciano Martelotto

[STEPS]
SECTION: Abstract
1. No nuclei left behind (NNLB)

The single-nuclei RNA sequencing protocol used by the Spatial Technologies Unit at the Beth Israel Deaconess Medical Center in order to isolate and sequence RNA from very small samples (<1000 cells). All steps are optimized to ensure maximum cell recovery.

This protocol has been successfully applied in order to perform single-nucleus RNA sequencing on isolated peripheral lymphatic vessels for HuBMAP.

This protocol has been developed in collaboration Luciano Martelotto at the University of Adelaide.
SECTION: Necessary materials
2. Very small pieces of tissue (~10-20 mg range. 1/2 - 1 grain of rice size)
EZ Lysis Buffer (NUC101-1KT, Sigma-Aldrich)
Pestle for 0.5 mL Eppendorf tube (Fisher Scientific, NC9719656)
DNA LoBind 0.5 mL and 1.5 mL Eppendorf tubes
High recovery PCR tubes
Swinging backet centrifuge (essential!l)
Protector RNAse Inhibitor (Sigma-Aldrich) or RiboLock RNAse Inhibitor (Thermofisher)
pluriStrainer Mini 70 μm Cell Strainer
pluriStrainer Mini 40 μm Cell Strainer
Flowmi Cell Strainers
SECTION: Buffers
3. Salty-Ez10 or Salty-Ez50 Lysis Buffer

10 mM Tris-HCl pH 7.5
146 mM NaCl
1 mM CaCl2
21 mM MgCl2
0.03% Tween-20 (Sigma Aldrich, P9416-50ML)
0.01% BSA (Miltenyi, 130-091-376)
1 mM DTT (Thermo)
10% Ez Lysis Buffer OR 50% Ez Lysis Buffer (Sigma Aldrich)
0.2-1 U/uL Protector RNAse Inhibitor

Wash and Resuspension Buffer
1x PBS
1% BSA (Molecular Grade)
0.2-1 U/uL Protector RNAse Inhibitor
SECTION: Protocol
5. Place small pieces of tissue in a pre-cooled 0.5 mL Eppendorf tube. Note: pieces can be fresh
or snap-frozen. Briefly spin the tube to consolidate the tissue at the bottom of the tube.
SECTION: Protocol
6. Add enough volume of chilled SaltyEZ10 (or SaltyEZ10) buffer just to cover the tissue.
SECTION: Protocol
7. Mice using the pestle. Perform at least 10 strokes or until the tissue is fully homogenized.
Then, add 200 uL more of chilled lysis buffer, and mix by pipetting 3x with a P1000 (bore tip is
optional).
SECTION: Protocol
8. Incubate the homogenized tissue on ice for 10’ to allow for nuclei release. Note: 10’ is usually
enough for most samples, yet it may require optimization to avoid over-lysing. I recommend
not to process more than 4 samples at the time because timing because difficult.
SECTION: Protocol
9. Filter homogenate using a 70 um-strainer mesh (pluriStrainer Mini 70 μm Cell Strainer) fitted
on a pre-cooled 1.5 uL Eppendorf. This step is to remove undigested tissue or fat prior to
centrifugation. Spin at 4°C for 10” to ensure all the volume is collected. Transfer the volume
to a pre-cooled 0.5 mL Eppendorf tube (~350 uL). Add 100 uL of lysis buffer to the 1.5 uL
Eppendorf to collect any leftover nuclei, wash walls and transfer onto the 0.5 mL Eppendorf
tube (~450 uL).
SECTION: Protocol
10. Centrifuge the nuclei at 500G for 5’ at 4°C to pellet the nuclei using a swinging bucket centrifuge. For this you will need to make an “0.5 mL adapter” if you don’t have 1.5 mL and 0.5 mL Eppendorf tubes enabled rotors. To do so, remove cap of a 5 mL and a 1.5 mL Eppendorf tubes, and insert the uncapped 1.5 mL tube inside the 5 mL tube.
SECTION: Protocol
11. Carefully remove the supernatant, leaving ~30 uL behind. Then resuspend the nuclei pellet by
adding 50 uL of chilled lysis buffer (~80 uL) and transfer the nuclei to a 0.2 uL PCR tube. Add
100 uL of lysis buffer to the 0.5 uL Eppendorf to collect any leftover nuclei, wash walls and
transfer onto the 0.2 mL PCR tube (~160 uL).
SECTION: Protocol
12. Centrifuge the nuclei at 500xg for 5’ at 4°C to pellet the nuclei using a swinging bucket
centrifuge. For this you will need to make an “o.2 mL adapter” removing the cap of a 0.5 mL
Eppendorf and insert the uncapped 0.5 mL tube in the 0.5 mL adapter made (step 6).
SECTION: Protocol
13. Wash 1: Carefully remove the supernatant, leaving ~30 uL behind. Very slowly, add 150 uL of Washing and Resuspension Buffer on the walls of the tube without resuspending the nuclei pellet (~180 uL).
SECTION: Protocol
14. Centrifuge the nuclei at 500xg for 5’ at 4°C to pellet the nuclei using a swinging bucket centrifuge.
SECTION: Protocol
15. Wash 2: Carefully remove the supernatant, leaving ~30 uL behind. Very slowly, add 150 uL of Washing and Resuspension Buffer on the walls of the tube without resuspending the nuclei pellet (~180 uL).
SECTION: Protocol
16. Carefully remove the supernatant, leaving ~50 uL behind and resuspend the pellet by gently pipetting 15x.
SECTION: Protocol
17. Count nuclei by using 5 uL of nuclei suspension and mixed 5 uL of Washing and Resuspension
Buffer (this is ½ dilution). If the nuclei prep is clumpy, add 100 uL Washing and Resuspension
Buffer and pass it through a 40 um Flowmi filter, then centrifuge 500xg for 5’ at 4°C, carefully remove the supernatant, leaving behind 50 uL. Resuspend by gently pipetting 15x (Note: this
step can cause significant loss).
SECTION: Protocol
18. Count and proceed to downstream single nuclei profiling.
SECTION: Important Notes
4. Salty-Ez10 is milder than Salty-Ez50, so choice Salty-Ez10 or Salty-Ez50 Lysis Buffer depends on the tissue and some testing will be required.
It is important to keep the tissue on ice at all times to prevent denaturation and degradation of the nuclei.
DNA LoBind Eppendorf tubes and Max recovery PCR tubes are essential to avoid loses.
Swinging bucket centrifuges are critical.