TITLE: Nuclei Extraction for tissue using Iodixanol Gradients
AUTHORS: Kriegstein lab

[DESCRIPTION]
Nuclei isolation using Iodixanol gradients geared for multiome

[STEPS]
SECTION: Creating Buffers
1. Stock Buffers
All stock solutions should be filtered using a .22 um PVDF/PES filter system. All solutions except 50% Iodixanol solution are stable at 4c for at least 6 months.

1 M Sucrose (300 mL)
Substance:                    Stock Conc.:        Amount:                Final conc. in working solution:
Sucrose                            -                            102.69 g                1 M
Water                                -                            235.5 mL                -

1.0616x Homogenization Buffer Stable Solution (200 mL)
Substance:                    Stock conc:            Amount:            Final conc. in working solution:
Sucrose                            1 M                        53.1 mL                .2653 M
KCl                                    2 M                        2.66 mL                26.6 mM
MgCl2                               1 M                        1.06 mL                5.31 mM
Tricine-KOH pH 7.8      .75 M                        5.67 mL                21.2 mM
Water                                    -                          137.5 mL                    -

Diluent Buffer (100 mL)
Substance:                    Stock conc:             Amount:            Final conc. in working solution:
KCl                                    2 M                            7.5 mL                150 mM
MgCl2                              1 M                             3 mL                    30 mM
Tricine KOH pH 7.8        .75 M                        16 mL                  120 mM
Water                                    -                             73.5 mL                    -

50% Iodixanol Solution (50 mL) **Remake Monthly for Stability 
Substance:                    Stock conc:              Amount:            Final conc. in working solution:
Diluent Buffer                        -                            8.3 mL                    -
Iodixanol                           60%                          41.7 mL                50%

1x Homogenization Buffer Unstable Solution for 4 reactions Prepare Fresh
Substance:                    Stock conc:              Amount:            Final conc. in working solution:
HB stable solution        1.0616X                    7536 uL                1X
DTT                                    1 M                            8 uL                    1 mM
Spermidine                     500 mM                      8 uL                    .5 mM
Spermine                        150 mM                      8 uL                    .15 mM
NP40                                10%                            240 uL                .3%
cOmplete PI                    100X                           80 uL                    1X
(diluted in HB stable) 
Ribolock                            40U/uL                      120 uL                .6 U/uL

30% Iodixanol Solution per reaction Prepare Fresh
Substance:                    Stock conc:                Amount:            Final conc. in working solution:
HB unstable                        -                                240 uL                        -
50% Iodixanol Solution    50%                            360 uL                    30%

40% Iodixanol Solution per reaction Prepare Fresh
Substance:                    Stock conc:                Amount:            Final conc. in working solution:
HB unstable                        -                                120 uL                        -
50% Iodixanol Solution    50%                            480 uL                    40%

Wash Buffer 1 mL for 4 reactions Prepare Fresh
Substance:                    Stock conc:                Amount:            Final conc. in working solution:
Tris-HCl pH 7.4                   1 M                            10 uL                    10 mM
NaCl                                      5 M                            2 uL                      10 mM  
MgCl2                                    1 M                            3 uL                        3 mM
BSA                                        30%                          33.3 uL                      1% 
Tween-20                              10%                          10 uL                        .1%
DTT                                        1 M                            1 uL                        1 mM
Ribolock                                40 U/uL                    15 uL                      .6 U/uL                           






SECTION: Creating Buffers
2. Before starting protocol:
1) pre-chill swinging bucket centrifuge and a fixed angle centrifuge to 4c
2) Pre-chill dounces and pestles to 4c on ice
3) Pre-chill tubes
4) Fill up 2 L beaker with 500 mL sterile water to soak the used Dounces
SECTION: Creating Buffers
3. Isolation of Nuclei via Dounce Homogenization and Density Centrifugation
1) place 20-50 mg frozen tissue or crushed into pre-chilled 7 mL dounce containing 1 mL cold 1x HB
2) Dounce with "A" loose pestle until resistance goes away (~10 strokes)
3) Dounce with "B" tight pestle until resistance goes away (~15 strokes)
4) Place "A" and "B" into sterile water to soak for cleaning later
5) Filter during transfer into FACS tube
6) Place Dounce into beaker with sterile water to soak for cleaning later
7) Pellet nuclei by spinning 5 min at 4c at 350 xg in a fixed angle centrifuge
8) Remove 950 uL of supernatant (50 uL remaining)
9) Gently resuspend nuclei in 350 uL 1x HB 
10) Add 1 volume (400 uL) of 50% Iodixanol solution and pipette mix
11) Slowly layer 600 uL of 30% Iodixanol solution under the 25% mixture. Wipe side of pipette tip with kimwipe to avoid mixing layers. 
12) Layer 600 uL 40% Iodixanol solution under the 30% mixture. Wipe side of pipette tip with kimwipe to avoid mixing layers.
13) In a pre-chilled swinging bucket centrifuge, spin for 20 min at 4c at 3000 xg with the brake off. Set acceleration level to 1 and deceleration at 0. (centrifuge time=23 min, time to stop=13 min)
14) Slowly extract top layers in increments of 200 uL down to 200-300 uL of nuclei band between 30% and 40% interface. 
15) Take 200 uL of the nuclei band and put into 1.5 mL LoBind tube
16) Dilute nuclei by adding 200 uL of wash buffer and mix by pipetting
17) Count nuclei using trypan blue
18) Centrifuge at 500 xg for 5 min at 4c
19) Remove supernatant without disrupting pellet
20) resuspend in x uL of chilled Nuclei Buffer (depending on what isolated nuclei are used for) to achieve target concentration based on count.