Blue:RNA-Seq Experiments:12052013
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Human Nuclei C1 Experiment: 12-05-2013[edit]
Purpose[edit]
- To generate single nuclei RNA-seq libraries using the C1 system on nuclei prepared from the human brain
- To compare NeuN stained versus unstained in RNA-seq library formation
FACS Sort[edit]
- DAPI stained or DAPI/NeuN stained (Note: Rnase Inhibitor was added during staining) human nuclei from Gwen (J. Chun Lab):
- Added ~1ul DAPI just prior to FACS sort on the Aria (Scripps)
- Nuclei were maintained on ice
- Added 50ul of sort buffer containing 0.5% FAF-BSA to low-bind 1.5ml eppendorf tube for collection
- Gated nuclei on DAPI positive and shifted gate slightly along the FSC axis to exclude the smallest nuclei
- Sorted 150,000 DAPI+ nuclei and 75,000 NeuN+ nuclei into separate collection tubes (final volume of ~1000ul)
- Note that the DAPI positive showed banding pattern. For NeuN stained we selected only the lowest band (single nuclei) and avoided the upper bands that could be multiples. This was not done for the DAPI+ alone
- Maintained on ice. Centrifuged 2000rpm at 4C for 10 minutes. Removed supernatant down to ~10ul. Resuspended pellet with ~20ul 0.5%BSA/PBS using p1000 by pipetting about 10 times.
- Counted nuclei using BioRad automatic cell counter: 1200 nuclei/ul for DAPI+ and 1100 nuclei/ul for NeuN+
- Adjusted [nuclei] to 750 nuclei/ul for both (final volume of 45-50ul)
- DAPI stained or DAPI/NeuN stained (Note: Rnase Inhibitor was added during staining) human nuclei from Gwen (J. Chun Lab):
C1 Loading[edit]
- Medium Chip was primed
- Nuclei and cells were mixed as followes:
- 5ul DAPI+
- 5ul NeuN+
- 5ul 0.5%BSA/PBS
- 10ul C1 Cell Suspension RGT
- Note: Final loading onto chip was 1500 nuclei
C1 Capture[edit]
- Each capture site was viewed using a regular inverted microscope.
- Capture Table:
Capture Site | # of DAPI | # of NeuN | Multiple |
1 | --- | 1 | --- |
2 | --- | 1 | --- |
3 | --- | 1 | --- |
4 | --- | 1 | --- |
5 | --- | 1 | --- |
6 | 1 | 1 | 2 |
7 | --- | --- | --- |
8 | --- | --- | 7 |
9 | 1 | --- | --- |
10 | --- | 4 | 4 |
11 | --- | 1 | --- |
12 | --- | 1 | --- |
13 | 1 | 1 | 2 |
14 | 2 | --- | 2 |
15 | 1 | 2 | 3 |
16 | --- | 1 | --- |
17 | 2 | 2 | 4 |
18 | 1 | --- | --- |
19 | --- | 1 | --- |
20 | --- | 1 | --- |
21 | 1 | 1 | 2 |
22 | 1 | --- | --- |
23 | --- | --- | --- |
24 | 7 | 1 | 8 |
25 | 1 | --- | --- |
26 | --- | --- | 10+ |
27 | --- | 1 | --- |
28 | 1 | 1 | 2 |
29 | --- | 1 | --- |
30 | --- | --- | 10+ |
31 | --- | --- | 10+ |
32 | --- | --- | 10+ |
33 | 1 | --- | --- |
34 | --- | --- | 10+ |
35 | --- | --- | 10+ |
36 | --- | --- | 6 |
37 | 1 | --- | --- |
38 | 1 | 1 | 2 |
39 | --- | --- | 10+ |
40 | 1 | 1 | 2 |
41 | --- | --- | 9 |
42 | --- | 1 | --- |
43 | 3 | 2 | 5 |
44 | --- | --- | 10+ |
45 | --- | --- | 10+ |
46 | --- | --- | 10+ |
47 | --- | --- | 10+ |
48 | --- | --- | 10+ |
49 | --- | --- | --- |
50 | 1 | --- | --- |
51 | --- | 1 | --- |
52 | --- | 1 | --- |
53 | --- | 1 | --- |
54 | --- | --- | --- |
55 | --- | 1 | --- |
56 | --- | --- | 6 |
57 | --- | 1 | --- |
58 | --- | 1 | --- |
59 | 1 | --- | --- |
60 | 1 | --- | --- |
61 | --- | 1 | --- |
62 | 1 | --- | --- |
63 | 1 | 1 | 2 |
64 | --- | --- | --- |
65 | --- | 1 | --- |
66 | --- | 1 | --- |
67 | --- | --- | --- |
68 | 1 | --- | --- |
69 | --- | --- | --- |
70 | --- | 1 | --- |
71 | --- | 1 | --- |
72 | --- | 1 | --- |
73 | 1 | --- | --- |
74 | --- | --- | --- |
75 | --- | --- | --- |
76 | --- | --- | 5 |
77 | --- | --- | --- |
78 | 1 | --- | --- |
79 | --- | --- | --- |
80 | 1 | --- | --- |
81 | --- | 1 | --- |
82 | --- | --- | --- |
83 | --- | 1 | --- |
84 | 2 | --- | 2 |
85 | --- | 1 | --- |
86 | --- | --- | --- |
87 | --- | --- | --- |
88 | --- | --- | --- |
89 | --- | --- | --- |
90 | --- | 1 | --- |
91 | --- | --- | --- |
92 | --- | --- | --- |
93 | --- | 1 | --- |
94 | 1 | 1 | 2 |
95 | --- | --- | --- |
96 | --- | 1 | --- |
C1 Run[edit]
- ERCC stock (1:100 dilution aliquot from original stock) was diluted 1:20 in C1 loading buffer for final of 1:2000 dilution added to lysis buffer
- Time from FACS sort to running C1 = ~3hours
Results[edit]
- 1500 Nuclei
- 80% Captured (19 empty chambers)
- 47% Single Capture
- Single DAPI+ = 14
- Single DAPI+NeuN+ = 31
- Number of clusters greater than or equal to 10 = 12. These were primarily located closer to the input chamber #48.
- Again the use of BSA allowed a much greater yield following sort and centrifugation. 1500 nuclei appears to be close to the optimal number for medium chip loading
- Its interesting that the NeuN stained showed almost three times the number of single nuclei captured and that during the sort we noticed that the NeuN+ were 1/3 of the total number of DAPI positive nuclei. This suggests that the medium chip size might be preferentially capturing the larger neuronal nuclei and as such would require more input for non-NeuN stained do to the smaller non-neuronal cell types within the pool. Put another way - the medium chip might have a selection bias for the larger neuronal nuclei.