Blue:RNA-Seq Experiments:09302013: Difference between revisions
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== Purpose == | == Purpose == | ||
*mNPCs from E12.5 mice | *mNPCs from E12.5 and E10.5 mice were isolated by Yun | ||
*mNPCs (mixed population of | *Sorting of mNPCs (mixed population of cell types) was based on PI negative staining | ||
== Smart-Seq Sort Buffer: == | == Smart-Seq Sort Buffer: == | ||
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| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Clontech Dilution Buffer|| || || | | Clontech Dilution Buffer|| || ||820ul | ||
|- | |- | ||
| Rnase Inhibitor||40U/ul||1U/ul|| | | Rnase Inhibitor||40U/ul||1U/ul||21.25ul | ||
|- | |- | ||
| ERCC spike-in RNA||1:10^5|||| | | ERCC spike-in RNA||1:10^5||||8.5ul | ||
|- | |- | ||
| total|||||| | | total||||||850ul | ||
|- | |- | ||
| | | | ||
Line 26: | Line 25: | ||
::'''Note: using 2ul per well for 4 plates | ::'''Note: using 2ul per well for 4 plates (two E12.5 and two E10.5) | ||
== Universal Sort Buffer: == | == Universal Sort Buffer: == | ||
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| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| | | DEPC Water|| || ||469ul | ||
|- | |- | ||
| | | 10% Triton-X100|| || ||5ul | ||
|- | |- | ||
| | | Rnase Inhibitor||40U/ul||1U/ul||12.5ul | ||
|- | |- | ||
| total|||||| | | ERCC spike-in RNA||1:10^5||||13.25ul | ||
|- | |||
| total||||||500ul | |||
|- | |- | ||
| | | | ||
Line 50: | Line 50: | ||
::'''Note: using 0.75ul per well for | ::'''Note: using 0.75ul per well for 5 plates (three for E12.5, two for E10.5) and bubbling just prior to sort | ||
== Plate Layout: == | == Plate Layout: == | ||
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| align="center" style="background:#f0f0f0;"|'''12''' | | align="center" style="background:#f0f0f0;"|'''12''' | ||
|- | |- | ||
| A|| | | A||Sc(1)||UHRR(9)||NC(17)||Sc(25)||UHRR(33)||NC(41)||Sc(49)||UHRR(57)||NC(65)||Sc(73)||UHRR(81)||NC(89) | ||
|- | |- | ||
| B||Sc(2)||Sc(10)||Sc(18)||Sc(26)||Sc(34)||Sc(42)||Sc(50)||Sc(58)||Sc(66)||Sc(74)||Sc(82)||Sc(90) | | B||Sc(2)||Sc(10)||Sc(18)||Sc(26)||Sc(34)||Sc(42)||Sc(50)||Sc(58)||Sc(66)||Sc(74)||Sc(82)||Sc(90) | ||
Line 97: | Line 95: | ||
'''Notes:''' | '''Notes:''' | ||
*Sort was done on the FACS ARIA II | *Sort was done on the FACS ARIA II | ||
*Sort files: [[File:9-30-13_E10.5_mNPC_snigle_cell_sort.pdf]] |
Latest revision as of 21:00, 30 September 2013
Purpose[edit]
- mNPCs from E12.5 and E10.5 mice were isolated by Yun
- Sorting of mNPCs (mixed population of cell types) was based on PI negative staining
Smart-Seq Sort Buffer:[edit]
Component | Stock | Final (1x) | Volume |
Clontech Dilution Buffer | 820ul | ||
Rnase Inhibitor | 40U/ul | 1U/ul | 21.25ul |
ERCC spike-in RNA | 1:10^5 | 8.5ul | |
total | 850ul | ||
- Note: using 2ul per well for 4 plates (two E12.5 and two E10.5)
Universal Sort Buffer:[edit]
Component | Stock | Final (1x) | Volume |
DEPC Water | 469ul | ||
10% Triton-X100 | 5ul | ||
Rnase Inhibitor | 40U/ul | 1U/ul | 12.5ul |
ERCC spike-in RNA | 1:10^5 | 13.25ul | |
total | 500ul | ||
- Note: using 0.75ul per well for 5 plates (three for E12.5, two for E10.5) and bubbling just prior to sort
Plate Layout:[edit]
' | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | Sc(1) | UHRR(9) | NC(17) | Sc(25) | UHRR(33) | NC(41) | Sc(49) | UHRR(57) | NC(65) | Sc(73) | UHRR(81) | NC(89) |
B | Sc(2) | Sc(10) | Sc(18) | Sc(26) | Sc(34) | Sc(42) | Sc(50) | Sc(58) | Sc(66) | Sc(74) | Sc(82) | Sc(90) |
C | Sc(3) | Sc(11) | Sc(19) | Sc(27) | Sc(35) | Sc(43) | Sc(51) | Sc(59) | Sc(67) | Sc(75) | Sc(83) | Sc(91) |
D | Sc(4) | Sc(12) | Sc(20) | Sc(28) | Sc(36) | Sc(44) | Sc(52) | Sc(60) | Sc(68) | Sc(76) | Sc(84) | Sc(92) |
E | Sc(5) | Sc(13) | Sc(21) | Sc(29) | Sc(37) | Sc(45) | Sc(53) | Sc(61) | Sc(69) | Sc(77) | Sc(85) | Sc(93) |
F | Sc(6) | Sc(14) | Sc(22) | Sc(30) | Sc(38) | Sc(46) | Sc(54) | Sc(62) | Sc(70) | Sc(78) | Sc(86) | Sc(94) |
G | Sc(7) | Sc(15) | Sc(23) | Sc(31) | Sc(39) | Sc(47) | Sc(55) | Sc(63) | Sc(71) | Sc(79) | Sc(87) | Sc(95) |
H | Sc(8) | Sc(16) | 10cell(24) | Sc(32) | Sc(40) | 10cell(48) | Sc(56) | Sc(64) | 10cell(72) | Sc(80) | Sc(88) | 10cell(96) |
Sc=Single cell; (#)=Indx
Notes:
- Sort was done on the FACS ARIA II
- Sort files: File:9-30-13 E10.5 mNPC snigle cell sort.pdf