Droplet capture of single nuclei, cDNA sequencing and data analysis

1.       Cut tissues into small pieces (1-2 mm) and incubate in RNAlater (ThermoFisher, cat# AM7021) overnight at RT

2.       Remove excess RNAlater and freeze tissue pieces in a sterile microfuge tube on dry ice. Store at -80oC until use

3.       On day of 10x run:  prepare homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris, pH 8.0, 1 µM DTT, 0.1% Triton X-100 (v/v).  and cool on ice.  The buffer can be prepared ahead of time and store at 4oC for up to 6 months, except for the DTT and Triton X-100.  Add those fresh on day of use.

4.       Cool down all necessary equipment and solutions

5.       Homogenize tissues in glass dounce homogenizer (Fisher Scientific, Cat# 357538) in 1ml of cold homogenization buffer (make fresh) 5 strokes with “loose” pestle and ~15-20 strokes with “tight” pestle … on ice

6.       Filter through 40 µm cell strainer (ThermoFisher Scientific, cat# 08-771-1)

7.       Transfer to microfuge tube (low bind; Sorenson BioScience, cat# 11700) and spin @ ~800g at 4o for 8 mins

8.       Remove supernatant and resuspend pellet in 500 µl of PBS + 1% BSA + SUPERaseIn RNase Inhibitor ( 0.2 U/µ; ThermoFisher Scientific, Cat# AM2696)

9.       Incubate on ice for 10-15 mins

10.   Add rabbit polyclonal anti-NeuN antibody (Millipore, cat#ABN78) 1:2000 to 1:5000

11.   Incubate with rotation at 4o for 30 mins

12.   Spin @ ~800g at 4o for 8 mins

13.   Remove supernatant and “wash” by adding 1 ml PBS + 1% BSA + SUPERaseIN RNase inhibitor

14.   Spin @ ~800g at 4o for 8 mins

15.   Resuspend pellet in 80 µl of PBS + 0.5% BSA + 2mM EDTA

16.   Add 20 µl of anti-rabbit IgG Microbeads (Miltenyi Biotech, cat# 130-048-602)

17.   Incubate at 4o 15-20 mins

18.   “wash” by adding 1 ml of PBS + 0.5% BSA + 2mM EDTA

19.   Spin @ ~800g at 4o for 8 mins

20.   Load onto LS column (Miltenyi Biotec, cat# 130-042-401) – follow instruction from manufacturer:

a.       put column on magnetic MidiMACS separator (Miltenyi Biotec, cat#130-042-302; MACS MultiStand (cat#130-042-303), add 3 ml of buffer (PBS + 0.5% BSA + 2mM EDTA) to equilibrate

b.       resuspend nuclei pellet (from above) in 0.5-1 ml buffer

c.       Add nuclei to column

d.       Wash column with 3 ml buffer (3x)

e.       Remove column from magnet and elute in 5 ml of same buffer

21.   Spin @ 500g at 4o for 10 mins

22.   Remove supernatant

23.   Add 1.5 ml of PBS + 1% BSA

24.   Homogenize with Ultra-Turrax (Laboratory Supply Network, Inc., cat#IKA:3737001) on setting 1 for 45 sec on ice (adjust time accordingly … depending on many nuclei you have)

a.       1st count:  Stain 10 µl with trypan blue and count (using a hemocytometer) also check for clumps

25.   Transfer to microfuge tube and spin @ ~800g at 4o for 8 mins

26.   Remove supernatant and resuspend in desire volume with PBS + 1% BSA … volume based on the first count. 

27.   Usually I try to go for 1000 nuclei/µl (if there are enough nuclei).  I also assume that the first count is not that accurate (too diluted), so usually resuspend nuclei in ½ the calculated volume.

28.   Stain 10µl with trypan blue and do a 2nd count

29.   Optional:  do a 3rd count.  This should be close to the 2nd count.

30.   **Homogenization Buffer:

31.   ** Can also use EZ PREP buffer (Sigma, Cat #NUC-101) for the homogenization buffer.

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