TITLE: Nuclei-seq
AUTHORS: Mandy Xu, Troy Ketela

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been developed by the Princess Margaret Genomics Centre specifically for Nuc-Seq, both at the bulk and single-cell levels. </div></div>

[STEPS]
?. [Nuclei-seq: Preparation]
Rack 4 falcon tubes (lysis buff, wash buff, nuclei, intermediate) and 1 FACS tube on ice
?. [Nuclei-seq: Preparation]
Chill centrifuges (4°C 800 g 10 min), clean hood, fill Styrofoam boxes: one with dry ice (petri dish and razor blade); one with ice packed and slanted for petri dish away from the user; one with ice with falcon tubes (above), 1 weighed epp tube and douncer with A pestle; Clean douncer, pestle and forceps with ethanol, wrap forceps and ethanol again; Pestle B and forceps on kimwipe beside ice in hood; Ranin wide bore tips and p1000;
?. [Nuclei-seq: Preparation]
Prep 5 epi tubes on a rack by scope: Three for checking the completion of lysis; One for final resuspension for FACs " leave on ice; One for counting.
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Weigh tissue (30-50mg ideal, save rest) and cut tissue into 1-2mm3 using a chilled razor on dry ice
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Add lysis buffer (enough to cover) and transfer to the ice to continue chopping, using a pipette to rinseoff blade using the lysis buffer of the sample
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Transfer to nuclei tube and lysis for 5 min
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Dounce supernatant first (1-2 strokes) and transfer to 4th tube
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Dounce pellet in thirds (2 strokes)
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Add enough lysis buffer to resuspend pellet in douncer, dounce and transfer to 4th tube
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Douncing is done when solution appears milky
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Resuspend 4th tube with wide bore tip and check with residual on tip and tip of p20 in SYBRusing countess chips
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Transfer super to nuclei tube, leaving behind thepellet
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Transfer pellet back to douncer and dounce with pestle B (1/3-1/2 of pellet depending ontoughness) " 2-4 strokes
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Rinse out douncer with lysis buffer
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Transfer to nuclei tube
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Resuspend nuclei and count again with leftover in p1000 tip " dounce again based on results
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Spin at 800g for 10 min
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Wash with 2ml of wash buffer
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Spin at 800g for 10 min
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Wash again with 2ml of wash buffer
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Spin for 10 min at 800g and resuspend in 1ml
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Filter with 40um Flowmi cell strainer, transfer to 1.5ml LoBind tube on ice
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Count nucleiSpin at 800g for 10 min
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Wash again with 2ml of wash buffer
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Spin for 10 min at 800g and resuspend in 1ml
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Filter with 40um Flowmi cell strainer, transfer to 1.5ml LoBind tube on ice
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Count nuclei
?. [Nuclei-seq: Nuclei Isolation from Tissue]
40ul buff + 5ul nuclei If >1 million use 10ul of DAPI If  DAPI requires 5 min incubation
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Make note of nuclei condition, number of strokes, nuclei size etc after loading
?. [Nuclei-seq: Nuclei Isolation from Tissue]
DO NOT DISCARD THE WASH AND RESUSPENSION BUFFER, KEEP IT ON ICE.
?. [Nuclei-seq: Nuclei Isolation from Tissue]
After FACS, Spin at 800g for 10 min, resuspend the pellet in cold wash and resuspension buffer (volume calculated so that the final conc is around 1000nuclei/ul)
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Count and take images
?. [Nuclei-seq: Nuclei Isolation from Tissue]
Make note of nuclei condition, number of strokes, nuclei size etc after loading
?. [Flow Sorting]
Stain the nuclei with DAPI at the concentration suggested by the manufacturer.
?. [Flow Sorting]
Cell sorter: Influx BRV (TMDT, LAB 2-504), sorting for 1-1.5hrs.
?. [Flow Sorting]
Gating: DAPI positive, and exclude any debris/ nuclei aggregates within the DAPI positive gating.
?. [Flow Sorting]
Collect nuclei in the Wash and Resuspension Buffer.
?. [Flow Sorting]
Wash the nuclei with the Wash and Resuspension Buffer.
?. [Flow Sorting]
Count the nuclei and proceed to 10X 3’ RNA seq directly.
?. [Flow Sorting]
After FACS, Spin at 800g for 10 min, remove the supernatant (sheath buffer+ wash and resuspension buffer), resuspend the pellet in a fresh, cold wash and resuspension buffer (volume calculated so that the final conc is around 1000nuclei/ul)Stain the nuclei with DAPI at concentration suggested by the manufacturer.