eamag/protocols-with-steps huggingface dataset:
TITLE: UC Davis - Immunohistochemistry IBA1
AUTHORS: Jennifer  Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Ionized calcium-binding adapter molecule 1 (IBA1) is specifically expressed in macrophages / microglia and is upregulated during the activation of these cells. Iba1 expression is up-regulated in microglia following nerve injury,[4] central nervous system ischemia, and several other brain diseases. Furthermore it has been found in athlesclerotic plaques and at sigts of vascular injury. </div><div class = "text-block"><span style = "font-weight:bold;">Modified from</span><span>: IHC Methods and Materials VMTH - Anatomic Pathology, UC-Davis</span></div></div>

[STEPS]
?. Immunohistochemistry was performed on four-micron thick, formalin-fixed, paraffin-embedded tissue sections, mounted on charged slides, and air-dried overnight at 37ºC.
?. Sections were deparaffinized through xylene to 100% reagent alcohol, and then treated with 0.3% hydrogen peroxide in 100% methanol for 30 minutes.
?. Sections were rehydrated to deionized water through 95% and 70% reagent alcohols. Antigen retrieval was performed on sections for IBA-1 with heat induced epitope retrieval in a Black & Decker Steamer using Target Retrieval Solution for 30 minutes at 95ºC, followed by a 20 minute cool down.
?. After antigen retrieval, slides were rinsed in deionized water and placed in 0.1M Phosphate Buffered Saline, pH 7.4 (PBS).
?. Sections were blocked for 20 minutes with antibody diluent and primary antibodies were applied without rinsing and incubated for 1 hour.   a. All post-antigen retrieval incubations are in a humidity chamber at room temperature.
?. After primary incubation, samples are rinsed twice for three minutes with PBS-Tween 20 between
each subsequent reagent application.
?. A single step, polymer based HRP (BioCare Medical, RC542H) was applied for 30 minutes to
label rabbit anti-IBA-1.
?. All labels were visualized with NovaRed for peroxidase (Vector SK-4800), per manufacturer’s
instructions.
?. Sections are counterstained in Mayer’s Hematoxylin, air dried and coverslipped.
TITLE: Preparation of fresh paraformaldehyde for mouse perfusion and tissue fixation
AUTHORS: thnasko

[DESCRIPTION]
Hnasko lab - preparation of fresh PFA for ms perfusion and tissue fixation

[STEPS]
1. 1.	For 1 Lof 4% paraformaldehyde, warm up 700 mLof ddH2O in a glass beaker using microwave until 40-60 °C.

2. 2.	Place on heated stir plate in a chemical fume hood, add a magnetic stir bar and start stirring and insert thermometer into solution.
3. 3.	Weigh 40 g of granular paraformaldehyde (PFA, EMS, cat# 19210).

4. 4.	Add PFA to the heated ddH2O solution and place on a stir plate in a chemical fume hood. Insert thermometer. Stir and heat to approximately 60 °C (paraformaldehyde flash point = 71°C). Avoid overheating.

5. 5.	The powder will not immediately dissolve into solution. Slowly raise the pH by adding about 1 mL NaOH 10N until the solution clears.

6. 6.	Once the paraformaldehyde is dissolved, add 100 mL 10X PBS (Fisher bioreagents, cat# BP399-1).

7. 7.	Cool solution to Room temperature before adjusting pH, can place on ice to speed cooling.

8. 8.	Adjust pH to ~pH 7.4 with small amounts of 12N HCl.

9. 9.	Adjust the volume of the solution to 1 L with ddH2O.

10. 10.	Filter the solution using a vacuum filtration flask (ThermoScientific, cat# 567-0020). Label and store in sealed container at 4 °C.
TITLE: Glycine-HCl Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Glycine-HCl Buffer]
Mix  and indicated volume of hydrochloric acid.  ABCDEFGHI1mL of HCl44.032.424.216.811.48.26.43.62pH2.22.42.62.83.03.23.43.6
pH range:   to   (a) 0.1 M Glycine: 7.5 g L-1 (M.W.: 75.0 g mol-1)(b) 0.1 M Hydrochloric acid
[glycine]
ABCDEFGHI1mL of HCl44.032.424.216.811.48.26.43.62pH2.22.42.62.83.03.23.43.6
?. [Glycine-HCl Buffer]
Mix and adjust the final volume to  with deionized water.
100 ml
?. [Glycine-HCl Buffer]
Adjust the final pH using a sensitive pH meter
TITLE: Th2 Polarization of Mouse CD4+ Cells
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Isolation of CD4+ Cells From Lymph Nodes]
Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
?. [Isolation of CD4+ Cells From Lymph Nodes]
Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in complete RPMI containing 10% FCS (complete medium).
?. [Isolation of CD4+ Cells From Lymph Nodes]
Resuspend cells in complete medium and use your favorite method to isolate CD4+cells. Consider using our Mojosort™ Mouse CD4 T Cell Isolation Kit.
?. [Th2 Polarization of CD4+ Cells]
On day 0, plate CD4+cells at 30 x106/30 ml/T-75 flask. Culture cells for 3 days in complete RPMI containing 10% FCS, Con A (5µg/mL), recombinant mouse IL-2 (20ng/ml), and recombinant mouse IL-4 (50ng/ml).
?. [Th2 Polarization of CD4+ Cells]
On day 3, harvest the cells and wash cells once. Seed 15 x106cells/30 ml/T-75 flask along with recombinant mouse IL-2 (20ng/ml) and recombinant mouse IL-4 (50ng/ml).
?. [Th2 Polarization of CD4+ Cells]
On day 5, coat a 60 x 15 mm tissue culture petri dish with anti-mouse CD3ε, clone 145-2C11, 10µg/mL in PBS, 5ml/dish. Incubate in a 4°C refrigerator overnight.
?. [Th2 Polarization of CD4+ Cells]
On day 6, wash the anti-mouse CD3ε pre-coated tissue culture petri dish with PBS. Harvest the cells from the flask (that were seeded on Day 5), wash them twice, and seed at 20 x106/10 ml/petri dish along with 10µl of monensin (1000x) and anti-mouse CD28, clone 37.51 (5µg/ml). Incubate for 6 hours at 37°C in a CO2 incubator.
?. [Th2 Polarization of CD4+ Cells]
After harvesting, the cells are ready for staining.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Forty SD male rats were divided into five groups randomly (n = 8 per group): the normal control (N) group, the normal saline group (NS), the light osteoarthritis group (A), the mild osteoarthritis group (B) and the heavy osteoarthritis group (C). On day 0, the rats in the A, B and C group were injected with 0.3, 1, 3 mg MIA respectively, and rats in the NS group were injected with 50 μl of saline solution.</p>
<p>The tissues at the acupoints Yanglingquan (GB34), Heding (EX-LE2) and Weizhong (BL40) were dissected. The dimensions of each tissue fragment were 1.5 × 1.5 × 1.5 mm. The tissues were fixed in 10% neutral formalin, dehydrated, embedded in paraffin and sectioned with a 4-μm slicer. Subsequently, the sections were dewaxed, dehydrated and stained in 0.5% toluidine blue for 30 min and then washed with tap-water.</p>
<p>the knee joints of 6 rats in each group were fixed in 10% neutral formalin after samples of GB34, EX-LE2 and BL40 were dissected. Then, the knee joints were placed in 5% formic acid for demineralization, dehydrated, embedded in paraffin and sectioned with a 5-μm slicer. Subsequently, the sections were dewaxed, dehydrated, and stained in 1% fast green for 1.5 min and then differentiated by acetic acid. Next, the sections were stained by 0.5% safranine-O for 1.5 min, differentiated by ethanol, and washed with tap-water.</p>
<p> The tissues at GB34, EX-LE2, and BL40 were dissected, fixed in 4% paraformaldehyde for 2.5 h, and dehydrated by 25% sucrose for 2 days. A frozen 10-μm section was sliced by a freezing microtome. For staining, the sections were washed in 0.1 M PB (pH=7.4) and blocked in 0.1 M PB (pH=7.4) containing 3% normal donkey serum and 0.5% Triton X-100 for 30 min. Next, the sections were treated with mouse monoclonal mast cell tryptase antibody and goat polyclonal serotonin antibody and incubated overnight at 4℃. After PB washes, the sections were exposed to Donkey Anti-Mouse IgG Alexa Fluor 488 and Donkey Anti-Goat IgG Alexa Fluor 594 for 2 hours. Subsequently, the sections were washed with PB and stained with DAPI for 6 minutes. After washing with PB, the sections were observed under a confocal laser scanning microscope. Double immunohistochemical staining using the same staining protocol was undertaken, to examine the co-expression of mast cell tryptase and HA.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: UV Exposure Protocol
AUTHORS: Martin O Pollard

[DESCRIPTION]
A procedure utilising a UV crosslinker to expose isolated DNA to UV radiation with the aim of inducing lesions in the DNA typical of that kind of damage induced by UV.

[STEPS]
SECTION: UV treatment
1. Pipette 2.2 µL  of   which has a measured concentration of 227 µg/ml from stock tube to sterile plastic petri dish.
SECTION: UV treatment
2. Dilute material in petri dish with 22.8 µL of buffer to achieve a target of 25 µL of DNA at 20 ng/µl yielding 0.5 µg of DNA. Gently mix but do not agitate vigorously. 

SECTION: UV treatment
3. Expose tube to UV light using Crosslinker at 254 nm to exposing the DNA to 800 J/m2 (80000 μJ/cm2)
 

SECTION: UV treatment
3.1. Place sample in Crosslinker drawer
SECTION: UV treatment
3.2. Press the ENERGY button, enter the exposure energy of 80000 microjoules/cm2 as 80 on the keypad.
Note: YOUR ENERGY EXPOSURE SETTINGS DISPLAYED MUST BE MULTIPLIED BY 
100 to obtain the exposure in microjoules/cm2. If settings are correct, push ENTER on the touch pad. 
SECTION: UV treatment
3.3. Press START button to activate crosslinker and wait for cycle to finish.
SECTION: UV treatment
4. Transfer sample to eppendorf tube with pipette.
TITLE: PCR and gel electropheresis 
AUTHORS: Ashwinuday 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to confirm genes or DNA of interest from a template using PCR. Also to amplify required amount of genes to larger amounts.</div></div>

[STEPS]
?. Prepare the working stock solution of all the reagents required. If already prepared and stored, takeout from the refrigerator and thaw on ice.
?. Prepare the following mixes each of total  : -  Itemsnegative controltest sampleultrapure water (from Milli-Q)40 ul39 ulpfu Buffer (10X)5 ul (1X)5 ul (1X)Forward primer (20uM)1 ul (0.4uM)1 ul (0.4uM)Reverse primer (20uM)1 ul (0.4uM)1 ul (0.4uM)dNTPs (2.5mM)2 ul (0.1mM)2 ul (0.1mM)Pfu polymerase1 ul1 ulTemplate (100ng/ul)1 ul (100ng)-
50 µl
Itemsnegative controltest sampleultrapure water (from Milli-Q)40 ul39 ulpfu Buffer (10X)5 ul (1X)5 ul (1X)Forward primer (20uM)1 ul (0.4uM)1 ul (0.4uM)Reverse primer (20uM)1 ul (0.4uM)1 ul (0.4uM)dNTPs (2.5mM)2 ul (0.1mM)2 ul (0.1mM)Pfu polymerase1 ul1 ulTemplate (100ng/ul)1 ul (100ng)-
?. Perform PCR using a Thermo cycler with the following temperature settings (All temperatures are in degree Celcius) :TemperatureTime955 min9530 secs5540 secs722 min725 minSet the 2nd, 3rd and 4th steps to repeat 30 times (or maximum till 35)
TemperatureTime955 min9530 secs5540 secs722 min725 min
The PCR product can be immediately run analyzed using gel electrophoresis or can be stored in   or lower to be analyzed later.
?. To prepare the 1% agarose gel, mix  of agarose in  of 1X TAE buffer and heat well to form a clear solution.
All items which interact with the gel and EtBr should be handled with gloves. Also ensure, the gloved hands are not used to touch anything other than gel materials.
0.5 g
50 mL
Based on requirement higher amounts of gel can be prepared. So, accordingly larger combs and higher amount of EtBr can be used.
?. Then the mix is allowed to cool till it is bearable to touch. Carefully add  of EtBr to the mix and swirl well.
2 µl
?. Then into a set gel cast with the appropriate comb (which is decided by the number of wells and the size of the wells required), pour the mix without bubbles.
If there is any bubble remove it with a tip soon after pouring
?. Once the gel solidifies, carefully take it out with a gloved hand and place in the buffer tank with enough buffer to cover the immersed gel and remove the comb without breaking the wells.
?. Mix approximately  samples with 1X loading dye and load into each gel. Load a 1kb ladder (or any suitable ladder) into the first lane. Run the gel at 100V
10 µl
?. After the dye front crosses 75%of the gel length, stop the current.
?. Take the gel out (with gloved hands) by letting all the buffer out into the tank and then image the gel on UV.Compare the sample bands with the ladder to estimate the bands of interest
TITLE: Conventional fixation method for Tetrahymena  thermophila
AUTHORS: miao.tian  

[DESCRIPTION]
A rapid and robust method for fixing Tetrahymena thermophila cells

[STEPS]
SECTION: General description of the method
1. It works for the IF of cytoplasm, nucleoplasm, and chromatin-bound proteins, FISH staining of receptive sequences (e.g., telomeric repeats, TEs). Nuclear and cell morphology are nicely maintained.

It does not work for the IF of Cna1, γ-H2A.X, and tubulin.
SECTION: Reagents
2. -    37% Formaldehyde (! Toxic, handel it in a fume hood)
-    Triton X-100
-    Sucrose
-    Paraformaldehyde (!Toxic; Cat# A3813, 1000. Applichem)
SECTION: Recipes for making solutions
3. Preparing 4% formaldehyde/ 3.4% sucrose fixative 100 mL

SECTION: Recipes for making solutions
4. 10% Triton X-100 50 mL
-    Add 5 mL of Triton X-100 into 45 mL of water. Microwave for 5s will let Triton X-100 dissolve rapidly.

SECTION: Recipes for making solutions
3.1. Weight 4 g of Paraformaldehyde, add 100 mL of water.

-    Heat to dissolve Paraformaldehyde in a fume hood. Namely, placing the flask on top of a 170 °C heat plate with stirring function, for around 25 min, with stirring. Try not let it boil.
-    Leave it on bench to cool for around 30 min (do not make it completely cool, otherwise sugar dissolves very slow).

SECTION: Recipes for making solutions
3.2. Add 3.4 g of Sugar into the warm paraformaldehyde solution, stir/dissolve it.

-    leave it at room temperature overnight and then store it in a fridge.
SECTION: Fixation of 5 mL of Tetrahymena cells
5. (!) For growing cells: Wash cells once with 10 millimolar (mM) Tris-HCl (pH 7.5) and resuspend cells with the same volume of 10 millimolar (mM) Tris-HCl (pH 7.5). Starvation or conjugation cells need not to be washed for another time.

SECTION: Fixation of 5 mL of Tetrahymena cells
6. Shoot 250 µL of 10% Triton X-100 (first) and 500 µL of 37% formaldehyde into cells. Invert tube ~ 5 times and keep it at room temperature (25 °C) for 30 min;

SECTION: Fixation of 5 mL of Tetrahymena cells
7. Collect cells by centrifugation at 1500 rpm for 1 min and pour supernatant (! contains formaldehyde, think about waste disposal rules)

SECTION: Fixation of 5 mL of Tetrahymena cells
8. Resuspend cell pellet with ~ 500 µL of 4% formaldehyde/ 3.4% sucrose fixative, mixing cells by gentle pipetting is recommended.

SECTION: Fixation of 5 mL of Tetrahymena cells
9. Cells are ready to apply onto slides. After drying in a fume hood for 1hr, slides are ready for staining.
TITLE: gRNA design and cloning with SapI into Loop plasmid L2_lacZgRNA-Cas9-CsA
AUTHORS: Eftychis Frangedakis, marta tomaselli, Susana Sauret-Gueto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to design and clone the guide RNA target sequence into a L2 plasmid ready to accept the gRNA by cloning with SapI (L2 plasmid also contains a cassette to express Cas9)</div></div>

[STEPS]
?. [Protocol for design of gRNA and cloning into L2 with SapI]
gRNA oligo designOrder two oligos that contain the forward and reverse guide sequence plus the overhangs necessary for ligation (highlighted with bold) into L2_lacZgRNA-Cas9-CsA: oligo F: 5’-  TCG-NNNNNNNNNNNNNNNNNNNN-gt 3’oligo R: 5’-AAAac-NNNNNNNNNNNNNNNNNNNN-3’Note: Standard de-salted oligos are ok
?. [Protocol for design of gRNA and cloning into L2 with SapI]
Oligo annealingMix oligos with water as follow:oligo F (100μM)   1μloligo R (100μM) 1μl water  8μl Total volume  10μlAnneal in a thermocycler using the following parameters: 37oC for 30 min, 95oC for 5 min and then ramp down to 25oC at 5oC per min. After annealing the gRNA can be directly cloned into L2_lacZgRNACas9-CsA plasmid without the need of any further processing (step 4).
?. [Protocol for design of gRNA and cloning into L2 with SapI]
Cloning into backbone vector In a 0.2 mL tube set up the following reaction:  AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
  53  10x Tango buffer (Thermo Fisher)
  14  &nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)
    &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8L2_lacZgRNACas9-Csa (25-50 ng)19annealed oligo110  Final volume
  10 Place samples on the thermocycler and incubated using the following program: Assembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40. Incubate at 37 oC for 16 h. Confirm with sequencingAssembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40. Incubate at 37 oC for 16 h. Confirm with sequencing
AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
  53  10x Tango buffer (Thermo Fisher)
  14  &nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)
    &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8L2_lacZgRNACas9-Csa (25-50 ng)19annealed oligo110  Final volume
  10
?. [Summary of design of gRNA and cloning into L2 with SapI]
TITLE: Isolation of Adult Pig Ventricular Myocytes
AUTHORS: Robert  Harvey, Shailesh Agarwal

[DESCRIPTION]
This protocol is for the isolation of adult pig ventricular myocytes.

[GUIDELINES]
This protocol is in compliance with the Guide for the Care and Use of Laboratory Animals as adopted by National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the University of Nevada, Reno. All surgical procedures conducted by a licensed veterinarian. 

[STEPS]
1. Sedate adult pig (25 to 50 kg) with Telezol (500 mg, intramuscular). Administer atropine (1 mg, subcutaneous), to control salivary and bronchial secretions, and (200 mg, intravenous), to facilitate tracheal intubation. Anesthetize the animal with isoflurane (2-4%, inhalation). 
2. Shave the chest area and wash with betadine antiseptic solution. Check for loss of corneal and pain reflexes to ensure that the animal is in a deep anesthetic plane. Open the chest cavity by peeling back the skin over the left side of the chest wall and then making an incision between the 4th and 5th ribs. Use rib spreaders to hold open the incision.
3. Open the pericardium and remove the heart, placing it in ice cold cardioplegia solution containing (in mM) NaCl 130, KCl 27, MgSO4 1.2, KH2PO4 1.2, glucose 50, HEPES 6 (pH 7.4). Rinse the heart free of blood and transport to the laboratory in cardioplegia solution on ice. 
4. Dissect the heart, removing a section of the left ventricular free wall supplied by the left anterior descending (LAD) coronary artery. 
6. Ligate any leaking arteries at the periphery of the free wall in order to maintain perfusion pressure throughout  the bulk of the tissue. Continue perfusion using, constant pressure, until the effluent is free of any remaining blood (about 10 minutes).
7. Switch perfusion to Ca2+-free solution containing collagenase  type 2 (~200 U/ml) and protease type XIV (~1 mg/ml) for 20 minutes.
8. Stop enzyme digestion of the tissue by perfusing with enzyme-free KB solution containing (in mM) K-glutamate 110, KH2PO4 10, KCl 25, MgSO4 1.2, taurine 20, creatine 5, EGTA 0.5, glucose 20, HEPES 5 (pH 7.2) for 2 minutes.
9. Remove the tissue from the Langendorff apparatus and separate into different regions as necessary. The top half can be used to obtain myocytes from the base of the free wall, while the bottom half can be used to obtain myocytes from the apex.
10. Cut the tissue into small pieces, mince with scissors, and then filter through a 500 µm polyester mesh. Collect the filtrate containing isolated myocytes. Allow the cells to settle in KB solution maintained at 37°C for 25 minutes.
11. Aspirate the supernatant and then resuspend the pellet of isolated myocytes in Ca2+ containing solution for use in experiments.
5. Attache the ventricular free wall to a Langendorff apparatus by cannulating the proximal end of the LAD coronary artery, and perfuse the tissue with Ca2+-free solution containing (in mM) NaCl 140, KCl 5.4, MgCl2 2.5, glucose 11, HEPES 5.5 (pH 7.4) maintained at 37oC,.
TITLE: Nuclei isolation from  human brain cortex
AUTHORS: Xian Adiconis, Joshua Z Levin

[DESCRIPTION]
This protocol can be used on flash-frozen or RNAlater-preserved brain cortex. It is based on a previously published method (1) with a few modifications including, 1) Adding Recombinant RNase Inhibitor into the EZ prep lysis buffer in the incubation steps. 2) Passing the lysate through a 40 µm cell strainer after the tissue grinding. 3) Doing an additional wash. 4) Passing the nuclei suspension through a 20 µm filter before the final counting.

[STEPS]
1. Prepare the following lysis buffers, wash buffer and resuspension buffer, and keep on ice.
Lysis buffer1
 
Lysis buffer2
 
Wash buffer (PBS with 0.01% BSA and 0.04 U/µl RNase Inhibitor)
 
 Nuclei resuspension buffer (PBS with 1% BSA and 0.2 U/µl RNase Inhibitor)
 


2. Add 2 mLof cold Lysis buffer1 into an empty douncing tube and keep on ice.

3. Drop the frozen tissue into the douncing tube and submerge it in the lysis buffer. Start immediately grinding the tissue with pestle A 25 times or until resistance disappears. Continue grinding with pestle B 25 times. Pass the lysate through a 40 µm filter into a new 50 mL tube on ice.

4. Rinse the douncing tube with the remaining 2 mL Lysis buffer1, pass through the same filter and into the same 50 mL tube.

5. Mix the lysate well by inverting the tube 5 times and incubate on ice for 5 min.

6. Pellet the nuclei by 500 x g, 5 min, 4 °C. Carefully aspirate the supernatant and set the nuclei pellet on ice.

7. Resuspend the nuclei by adding 1 mLLysis buffer2 and mix well with a P1000 pipette. Add the remaining 3 mL Lysis buffer2, mix well by inverting, and incubate on ice for 5 min
8. Pellet the nuclei by 500 x g, 5 min, 4 °Cas in step 6. Carefully aspirate the supernatant and set the nuclei pellet on ice.
9. Resuspend the pellet with 750 µL Nuclei resuspension buffer, filter through a 20 µm filter, and collect into a 1.5 ml Eppendorf tube. Count the nuclei using Cellometer K2 (Nexcelom Bioscience) with the AO stain. Keep on ice until downstream processing.