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:

  1. Bulk unfixed and sorted human nuclei
  2. Bulk fixed and sorted Nuclei
  3. Bulk fixed and unsorted nuclei
  4. 300 fixed and sorted nuclei
  5. 300 fixed and sorted nuclei
  6. 150 fixed and sorted nuclei
  7. 150 fixed and sorted nuclei
  8. 30 fixed and sorted nuclei
  9. 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