Blue:RNA-Seq Experiments:09302013: Difference between revisions
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>B1lake (Created page with " == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | al...") |
>B1lake |
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(12 intermediate revisions by the same user not shown) | |||
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== Purpose == | == Purpose == | ||
* | *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: == | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Stock''' | |||
| | | align="center" style="background:#f0f0f0;"|'''Final (1x)''' | ||
|| | | align="center" style="background:#f0f0f0;"|'''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: == | ||
{| {{table}} | |||
: | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
| align="center" style="background:#f0f0f0;"|'''Stock''' | |||
| align="center" style="background:#f0f0f0;"|'''Final (1x)''' | |||
| align="center" style="background:#f0f0f0;"|'''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: == | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| | | align="center" style="background:#f0f0f0;"|'''2''' | ||
|||||| | | align="center" style="background:#f0f0f0;"|'''3''' | ||
| | | align="center" style="background:#f0f0f0;"|'''4''' | ||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| | | align="center" style="background:#f0f0f0;"|'''6''' | ||
| | | align="center" style="background:#f0f0f0;"|'''7''' | ||
| | | align="center" style="background:#f0f0f0;"|'''8''' | ||
| | | align="center" style="background:#f0f0f0;"|'''9''' | ||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''11''' | |||
| align="center" style="background:#f0f0f0;"|'''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]] |
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