TITLE: DNA Extraction from FANS sorted nuclei
AUTHORS: Stefania Policicchio,  Jonathan P Davies, Barry Chioza,  Aaron Jeffries,  Joe Burrage,  Jonathan Mill, Emma Dempster

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe an optimised protocol for the extraction of genomic DNA from frozen nuclei samples collected from different neural cells using Fluorescence-assisted Nuclei Sorting (FANS) of post mortem human brain. The same protocol is also suitable for DNA extraction from cells allowing optimal recovery in terms of DNA purity and yield.</div></div>

[STEPS]
?. [DNA extraction from FANS purified nuclei]
Defrost frozen FANS sorted nuclei contained in a 1.5ml eppendorf tube
on ice
NOTE 1-2 nuclei aliquots (~200,000 nuclei/tube) per each nuclei population should yield between 300-500ng DNA).
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To each sample add either 250µL / 500µL/ 1mL of Slagboom buffer (SB) depending on size of cell pellet. Normally 250µL is sufficient for 200,000 nuclei aliquot or 1x106 cells
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Add 500µL of SB to each nuclei sample (if nuclei pellet)*
* If nuclei are stored in running buffer: calculate the volume of 10x STE and 5% SDS to add to each sample to be consistent with the composition of SB (1x STE; 0.5% SDS).e.g. If you have collected a 400µL sample you would add 50µL 10x STE  and 50µL 5% SDS to make the sample up to 500µL
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Add 1µL of RNase A per 500uL buffer
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Incubate at  for  (using a heat block)
37 °C
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Add proteinase K to a final concentration 2mg/mL (e.g 5µL for every 500µL of SB)
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Mix by inverting 10 times (do not vortex or pipette mix)
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Incubate at  for  (water bath)
60 °C
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Move samples from the water bath and leave at  for
37 Room temperature
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Briefly spin down tubes (pulse spin)
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For every 500µL of SB used, add 100 µL of Majiik Mix (e.g. 200 µL for 1 mL of SB)
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Mix by vigorous inversions (DO NOT vortex)
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Centrifuge at  for  at
Centrifuge: 17000 34
37 Room temperature
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Carefully recover supernatant and transfer it to a new labelled 1.5ml tube (leaving ~50µL at the bottom of the tube)
?. [DNA extraction from FANS purified nuclei]
Repeat step above by adding another 100µL of Majiik Mix to the new tube
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Mix by vigorous inversions (DO NOT vortex)
?. [DNA extraction from FANS purified nuclei]
Centrifuge at  for
Centrifuge: 17000 34
37 Room temperature
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Recover supernatant and transfer to a new labelled 1.5ml tube leaving ~50ul at the bottom of the old tube.
NOTE - If supernatant exceeds 1mL, split into 2 tubes
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Add an equal volume of 100% Isopropanol to each tube (e.g. 1mL supernatant + 1 mL 100% Isopropanol)
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After adding the Isopropanol, add 0.7-1µL Glycogen Blue per tube (this step is optional – but advised as it helps to visualize DNA pellet at the end)
?. [DNA extraction from FANS purified nuclei]
Mix by inversion (10 times)
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Centrifuge at  for at  (hinges of tubes facing upwards)
Centrifuge: 17000 34
37 Room temperature
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Carefully remove supernatant and discard
NOTE - Care must be taken when pouring supernatant since the DNA pellet only weakly adheres to the side of the tube
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Proceed by adding 500µL of  80% ethanol to each tube
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Mix gently by pipetting
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Centrifuge at  for at
Centrifuge: 17000 34
37 Room temperature
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Carefully discard supernatant
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Pulse centrifuge to collect remaining liquid at the bottom of the tube
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Recover and discard residual liquid from the bottom of the tube making sure not to disturb the DNA pellet
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Leave DNA pellets to air dry for 5-15 minutes (leave lids open)
OPTIONAL - Leave to dry for additional   at   but be careful not to over-dry pellets
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Resuspend pellet in 20uL of ddH2O or 1xTE (15µL per tube if expected very low yield). Avoid pipette mixing, only gently flicking
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Pulse spin tubes to help pellet resuspend/ dislodge from the tube wall
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Leave tubes at  overnight to fully resuspend before quantifying DNA samples (Nanodrop or Qubit measurements).
4 °C