TITLE: Frozen Tissue Nuclei Extraction (v2)
AUTHORS: Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). </div><div class = "text-block"><span style = "font-weight:bold;">This protocol is based strongly on a similar extraction protocol from the </span><a href="http://mccarrolllab.org/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">McCarroll lab</span></a><span style = "font-weight:bold;">. </span></div></div>

[STEPS]
?. Make dissociation buffer, 50 mLs per sample:
?. Make extraction buffer,  3 mLs per sample: Dissociation Buffer + ​1%​ Kollidon VA64 + 1% Triton X100 + 1:40 RNase-inhibitor
?. Prepare equipment: Set out and chill the following equipment/supplies. The instructions below are for one sample.
   ● Cold block dissecting tray, kept at -20C
 
  ●  Chill clean razor blades and glass slides on the cold dissecting tray 

  ●  Chill 3 mL syringe with 26 1⁄2 gauge needle, at 4C, one syringe and needle per sample
  
  ●  Place a 12-well tissue-culture plate, well-bottoms colored with marker, on ice             * Color the underside of the culture plate with dark marker so you can see your sample dissociate  ●  Place 2 mL chilled extraction buffer in the first well of 12-well plate 
 
  ●  Place ~50 mL chilled DB in a falcon tube on ice
 
  ●  Locate DAPI (1:1000) (https://www.thermofisher.com/order/catalog/product/D1306) 
 
  ●  Locate 50 μm eppendorf tube filter, place in 4C
       (https://us.sysmex-flowcytometry.com/consumables/celltrics-filters/sterile-single-p ack-celltrics               filters/1445/sterile-single-pack-celltrics-filters-50/box?number=04-00 4-2327) 
 
  ●  Place 2 50 mL tubes on ice 
 
  ●  Locate a c-Chip FR hemocytometer
?. Chill all buffers to 4C, and keep all buffers on ice when in use.
?. Begin extraction. Perform all steps with cold buffers on ice. Nuclei should never leave the ice or 4C environment.Chop up tissue on the chilled dissection tray. If your sample is a very small piece (less than 3mm in any dimension) you can forgo this step and place sample immediately into the extraction bufferPlace the frozen sample on the glass slide and shave the frosty/freezer-burned tissue face you want to sample from off the sample and discard. This is damaged and not worth extracting. Then, moving rapidly but carefully, shave off “enough” sample to work with. 
 Rapidly mince the shaved sample with the 2 chilled razor blades that were used to shave the tissue block and scrape into the first well of a 6-well plate that contains 2 mL of cold extraction buffer. Pipette up and down 20 times.
?. Locate supplies for FACS:      ●  96 well cold block, chilled to -20C: 
        (https://www.daigger.com/eppendorf-pcr-coolers-14616-group?gclid=EAIaIQobCh 
        MI2rKM7aDN4AIVDRgMCh2jkwRMEAQYBSABEgKeA_D_BwE)
 
    ●  200uL 5% BSA-DB 
 
    ●  20uL collection buffer: DB with 1:40 RNAse-inhibitor          * Prepare the collection buffer right before beginning FACS session
?. Incubate in detergent with mechanical trituration: Commence 10-min incubation, pipetting up and down 20x every ~2.5 min with a 1000 uL pipette set to 1000uL.
?. Perform mechanical dissociation: Pull the sample into the chilled needle/syringe and express into the same well for a final mechanical dissociation.If large chunks remain, repeat this step, pulling the sample into the same syringe and expressing it into the same well.
?. 
?. Large volume wash: Extract the nuclei into empty 30ml tube. Add chilled DB to the extracted filtered nuclei to a total volume of just under 30 mL.
?. Split sample: Divide these diluted nuclei among 2 50 mL conical tubes (~15 mL per tube).
?. Spin tubes at 600 rcf in a swinging bucket centrifuge for 10 minutes at 4C.
?. Remove supernatant: Carefully aspirate ~13 mLs wash buffer with a serological pipette. To keep from disturbing pellet, gently aspirate the rest of the supernatant with a P1000 pipette. Leave about 500-300uL of sample in the bottom of the tube. Pipette sample to resuspend nuclei, being careful not to introduce bubbles. Pool the appropriate sample pairs back together.
?. Filter nuclei clumps: Wet the ​40um eppendorf tube filter with cold DB​, pass the ~0.5-1mL of washed sample through the filter into an epi tube.
?. Stain DNA: Add ​1ul of DAPI ​to 1ml of nuclei. Remove 20ul and place into FR hemocytometer. Check your nuclei concentration and nuclei quality. Some debris is ok.
?. FACS enrichment for singlets on singlet DAPI peak: We collect FACS'ed samples in PCR tubes held in a chilled (-20C) 96-well cold block to keep the collection volume as minimal as possible: (​https://www.daigger.com/eppendorf-pcr-coolers-14616-group?gclid=EAIaIQobChMI2rK M7aDN4AIVDRgMCh2jkwRMEAQYBSABEgKeA_D_BwE​)We FACS on a Sony SH800 sorter, using a 70um chip, with these settings:  
 
 We gate on the singlet DAPI peak:
?. Prepare the collection PCR tubes:
     ○ Fill the PCR tube with 5% BSA-DB, vortex, and remove BSA. This coats the sides of the tube so that if nuclei hit       the side of the tube they will fall to the bottom and not dry out.
     ○ Wash tube with 200 uL DB
     ○  Make collection buffer, add 20uL collection buffer (DB with 1:40 RNase-inhibitor) to the PCR tube. 
 FACS into the tube using a 70um chip with 1%FSC threshold on the DAPI peak.
?. Calculate nuclei concentration:After FACS, use a pipette to determine the volume in each tube. 
Make a 1:10 dilution of your sample: combine 18ul chilled DB with 2ul of nuclei a PCR tube to make a 1:10 dilution. Mix and put into FR hemocytometer. 
Visualize with the fluorescent scope, getting images of brightfield and DAPI-excited nuclei. Notice that debris is gone. 
 Count all 16 large squares to get the most accurate concentration estimate.Calculate the average of the 16 squares. Multiply that number by 10 (accounts for 1:10 dilution) then multiply by 5 (FC hemocytometer factor). This number is your final concentration in nuclei/uL.
?. Prep for 10X sequencing:The maximum nuclei you can input is 17000 (which comes out to 10000 after loss). For 10x v3 the input nuclei volume is 46.6, which means the maximum concentration you can input is 364 n/uL.