TITLE: Nuclei Isolation for SNARE-seq2 
AUTHORS: Carter Palmer, Christine Liu, Jerold Chun

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is intended to be used for the isolation of nuclei from fresh-frozen brain tissue in preparation for analysis by Single-Nucleus Chromatin Accessibility and mRNA Expression sequencing (SNARE-seq). It has been applied to tissues from mouse, marmoset, and human. </div></div>

[STEPS]
?. [Buffer Preparation]
Nuclei Extraction Buffer (NEB):  ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  Sucrose    320 mM         5.48g    Solid  3  Magnesium Acetate    3 mM         32.16mg    Solid  4  CaCl2    5 mM         50µl    5M Stock  5  EDTA    100 µM         10µl    0.5M Stock  6  Tris-HCl pH 8    10 mM         500µl    1M Stock  7  Triton X-100    0.1 %         500µl    10% Stock  8  &nbsp;MilliQ Water              Bring to 50ml       9  RNase Inhibitor    80 U/ml         100µl    40,000U/ml  10Protease Inhibitor Tablet            1 Tablet
ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  Sucrose    320 mM         5.48g    Solid  3  Magnesium Acetate    3 mM         32.16mg    Solid  4  CaCl2    5 mM         50µl    5M Stock  5  EDTA    100 µM         10µl    0.5M Stock  6  Tris-HCl pH 8    10 mM         500µl    1M Stock  7  Triton X-100    0.1 %         500µl    10% Stock  8  &nbsp;MilliQ Water              Bring to 50ml       9  RNase Inhibitor    80 U/ml         100µl    40,000U/ml  10Protease Inhibitor Tablet            1 Tablet
?. [Tissue Dissociation]
Remove tissue from -80oC storage and place on ice.
?. [Tissue Dissociation]
Image the tissue rapidly in front of a ruler
?. [Tissue Dissociation]
Add 1ml of ice cold NEB to the tissue and incubate on ice for
?. [Tissue Dissociation]
During the incubation, wash the homogenizer with MilliQ water, 10% bleach, 70% EtOH, and MilliQ water again
?. [Tissue Dissociation]
Rinse homogenizer with 1ml of ice cold NEB
?. [Tissue Dissociation]
Once the incubation is complete, add 1 ml of fresh NEB to the homogenizer and transfer the tissue and the 1ml of NEB it is in to the homogenizer
?. [Tissue Dissociation]
Homogenize the tissue using ~20 compressions with the pestle, or until the tissue is entirely dissociated, this step can be variable
?. [Tissue Dissociation]
Pass nuclei suspension through 50 micron filter into a 15 ml conical tube, wash filter with another 4ml of NEB
?. [Tissue Dissociation]
Wait for
?. [Tissue Dissociation]
Centrifuge the samples
Centrifuge: 820 34
?. [Tissue Dissociation]
Carefully aspirate the supernatant and slowly resuspend the pellet in 1ml of NEB
?. [Tissue Dissociation]
Gently add another 9 ml of NEB to the sample
?. [Tissue Dissociation]
Centrifuge the samples
Centrifuge: 820 34
?. [Nuclei Fixation]
Aspirate the supernatant and slowly resuspend the samples in 1ml of PBSE + Sucrose
?. [Nuclei Fixation]
Gently add another 5 ml of PBSE + Sucrose
?. [Nuclei Fixation]
Centrifuge the samples
Centrifuge: 820 34
?. [Buffer Preparation]
PBSE + Sucrose:  ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  Sucrose    320 mM         5.48g    Solid  3  EGTA    250 µM         25µl    0.5M   4  1x PBS pH 7.4              Bring to 50ml       5  RNase Inhibitor    40 U/ml         50µl    40,000U/ml  6Protease Inhibitor Tablet            1 Tablet
ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  Sucrose    320 mM         5.48g    Solid  3  EGTA    250 µM         25µl    0.5M   4  1x PBS pH 7.4              Bring to 50ml       5  RNase Inhibitor    40 U/ml         50µl    40,000U/ml  6Protease Inhibitor Tablet            1 Tablet
?. [Buffer Preparation]
PBSE + BSA:  ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  BSA    1%         500mg    Solid  3  EGTA    250 µM         25µl    0.5M   4  1x PBS pH 7.4              Bring to 50ml       5  RNase Inhibitor    40 U/ml         50µl    40,000U/ml  6Protease Inhibitor Tablet            1 Tablet
ABCDE1       Final Concentration         Chemical to Add    Stock Concentration  2  BSA    1%         500mg    Solid  3  EGTA    250 µM         25µl    0.5M   4  1x PBS pH 7.4              Bring to 50ml       5  RNase Inhibitor    40 U/ml         50µl    40,000U/ml  6Protease Inhibitor Tablet            1 Tablet
?. [Nuclei Fixation]
Aspirate all but approximately  of the PBSE+Sucrose, and gently resuspend the nuclei pellet in the remaining volume
100 µl
?. [Nuclei Fixation]
While gently vortexing, add 5ml of 0.5% Formaldehyde diluted in 1X PBS
?. [Nuclei Fixation]
Fix for 10 min on ice
?. [Nuclei Fixation]
Centrifuge the samples
Centrifuge: 820 34
?. [Nuclei Fixation]
Aspirate the supernatant and slowly resuspend the samples in 1ml of PBSE + Sucrose
?. [Nuclei Fixation]
Centrifuge the samples
Centrifuge: 820 34
?. [DAPI Staining and Sorting]
Aspirate the supernatant and slowly resuspend the samples in 1ml of PBSE + BSA
?. [DAPI Staining and Sorting]
Gently add another 10ml of PBSE + BSA
?. [DAPI Staining and Sorting]
Centrifuge the samples
Centrifuge: 820 34
?. [DAPI Staining and Sorting]
Make 1ml/sample of PBSE + BSA + DAPI by adding DAPI at a final concentration of 1.25 µg/ml to PBSE + BSA. This is a 1:4000 dilution from a DAPI stock at 5mg/ml
?. [DAPI Staining and Sorting]
Aspirate the supernatant and slowly resuspend the samples in 1ml of PBSE + BSA + DAPI
?. [DAPI Staining and Sorting]
Pass nuclei suspension through 50 micron filter into a FACS tube, gently tap on table to get all nuclei through the filter
?. [DAPI Staining and Sorting]
Use the DAPI singlet peak to sort singlet nuclei events into a 2ml low binding eppendorf tube.
?. [DAPI Staining and Sorting]
Store all samples on ice until SNARE-seq processing begins. Do not store for longer 4 hours before processing.