Blue:RNA-Seq Experiments:09232013
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Fluidigm C1 Run: Human Pyramidal Nuclei[edit]
- Samples prepared and sorted by Yun at Scripps (FACS ARIA)
- Bulk sorted nuclei (BD Influx) based on PI positive - ~120 nuclei/ul
- Centrifuged nuclei at 2000rpm 4C for 10 min (table-top centrifuge)
- Resuspended in low volume, added PI stain again to visualize nuclei and counted - ~250 nuclei/ul
- Used ERCC diluted 1:20,000 (10 fold less than previous experiment)
- Add 15ul of nuclei to 10ul cell suspension reagent and loaded 20ul onto chip (5ul actually loading into chip = 750 nuclei)
- Observed chip under microscope and counted occupancy rate:
Capture Site | # of Cells | Capture Site | # of Cells |
1 | 0 | 49 | 0 |
2 | 1 | 50 | 0 |
3 | 3 | 51 | 0 |
4 | 0 | 52 | 0 |
5 | 0 | 53 | 0 |
6 | 0 | 54 | 1 |
7 | 2 | 55 | 0 |
8 | 1 | 56 | 1 |
9 | 0 | 57 | 0 |
10 | 0 | 58 | 0 |
11 | 0 | 59 | 0 |
12 | 0 | 60 | 1 |
13 | 0 | 61 | 1 |
14 | 1 frag | 62 | 1 |
15 | 0 | 63 | 0 |
16 | 1 | 64 | 0 |
17 | 1 | 65 | 0 |
18 | 0 | 66 | 1 |
19 | 1 + 1 frag | 67 | 0 |
20 | 0 | 68 | 2 |
21 | 1 | 69 | 0 |
22 | 6 | 70 | 0 |
23 | 1 | 71 | 0 |
24 | 0 | 72 | 0 |
25 | 1 | 73 | 0 |
26 | 0 | 74 | 0 |
27 | 0 | 75 | 0 |
28 | 3 | 76 | 1 |
29 | 1 | 77 | 0 |
30 | 0 | 78 | 0 |
31 | 0 | 79 | 0 |
32 | 0 | 80 | 0 |
33 | 0 | 81 | 2 |
34 | 1 + 1 frag | 82 | 1 |
35 | 0 | 83 | 0 |
36 | 0 | 84 | 0 |
37 | 3 | 85 | 0 |
38 | 0 | 86 | 0 |
39 | 1 | 87 | 0 |
40 | 0 | 88 | 0 |
41 | 1 | 89 | 1 |
42 | 0 | 90 | 0 |
43 | 0 | 91 | 0 |
44 | 4 | 92 | 0 |
45 | 0 | 93 | 1 |
46 | 1 | 94 | 0 |
47 | 3 | 95 | 0 |
48 | 6 | 96 | 0 |
Plate Layout:
' | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | 3_C03 | 1_C02 | 0_C01 | 0_C49 | 0_C50 | 0_C51 | 0_C06 | 0_C05 | 0_C04 | 0_C52 | 0_C53 | 1_C54 |
B | 0_C09 | 1_C08 | 2_C07 | 0_C55 | 1_C56 | 0_C57 | 0_C12 | 0_C11 | 0_C10 | 0_C58 | 0_C59 | 1_C60 |
C | 0_C15 | 1f_C14 | 0_C13 | 1_C61 | 1_C62 | 0_C63 | 0_C18 | 1_C17 | 1_C16 | 0_C64 | 0_C65 | 1_C66 |
D | 1_C21 | 0_C20 | 1 + 1f_C19 | 0_C67 | 2_C68 | 0_C69 | 0_C24 | 1_C23 | 6_C22 | 0_C70 | 0_C71 | 0_C72 |
E | 1_C25 | 0_C26 | 0_C27 | 0_C75 | 0_C74 | 0_C73 | 3_C28 | 1_C29 | 0_C30 | 0_C78 | 0_C77 | 1_C76 |
F | 0_C31 | 0_C32 | 0_C33 | 2_C81 | 0_C80 | 0_C79 | 1 + 1f_C34 | 0_C35 | 0_C36 | 0_C84 | 0_C83 | 1_C82 |
G | 3_C37 | 0_C38 | 1_C39 | 0_C87 | 0_C86 | 0_C85 | 0_C40 | 1_C41 | 0_C42 | 0_C90 | 1_C89 | 0_C88 |
H | 0_C43 | 4_C44 | 0_C45 | 1_C93 | 0_C92 | 0_C91 | 1_C46 | 3_C47 | 6_C48 | 0_C96 | 0_C95 | 0_C94 |
Note: f=fragmented nuclei
RNA Extraction of Unfixed and Fixed Nuclei[edit]
Samples:
- Bulk unfixed and sorted human nuclei
- Bulk fixed and sorted Nuclei
- Bulk fixed and unsorted nuclei
- 300 fixed and sorted nuclei
- 300 fixed and sorted nuclei
- 150 fixed and sorted nuclei
- 150 fixed and sorted nuclei
- 30 fixed and sorted nuclei
- 30 fixed and sorted nuclei
- RNA extraction using the ZR RNA Microprep kit (with on column DNase treatment) and collected in 6ul
- Determined concentration for bulk samples using Qubit RNA kit - only detected RNA for the unfixed nuclei - fixation (1%paraformaldehyde) seemed to inhibit RNA extraction