Blue:RNA-Seq Experiments:09302013: Difference between revisions

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== Purpose ==
== Purpose ==


*mNPCs from E12.5 mice generated by Yun
*mNPCs from E12.5 and E10.5 mice were isolated by Yun
*mNPCs (mixed population of NSCs and more differentiated cortical neural cell types) were sorted based on PI negative staining
*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|| || ||772ul
| Clontech Dilution Buffer|| || ||820ul
|-
|-
| Rnase Inhibitor||40U/ul||1U/ul||20ul
| Rnase Inhibitor||40U/ul||1U/ul||21.25ul
|-
|-
| ERCC spike-in RNA||1:10^5||||8ul
| ERCC spike-in RNA||1:10^5||||8.5ul
|-
|-
| total||||||800ul
| total||||||850ul
|-
|-
|  
|  
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::'''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'''
|-
|-
| Clontech Dilution Buffer|| || ||384.7ul
| DEPC Water|| || ||469ul
|-
|-
| Rnase Inhibitor||40U/ul||1U/ul||10ul
| 10% Triton-X100|| || ||5ul
|-
|-
| ERCC spike-in RNA||1:10^5||||5.3ul
| Rnase Inhibitor||40U/ul||1U/ul||12.5ul
|-
|-
| total||||||400ul
| ERCC spike-in RNA||1:10^5||||13.25ul
|-
| total||||||500ul
|-
|-
|  
|  
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::'''Note: using 0.75ul per well for 4 plates and bubbling just prior to sort
::'''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||NC(1)||UHRR(9)||NC(17)||NC(25)||UHRR(33)||NC(41)||NC(49)||UHRR(57)||NC(65)||NC(73)||UHRR(81)||NC(89)
| 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)
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'''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: