Blue:RNA-Seq Experiments:07102013
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Purpose[edit]
- Sort single mouse NPCs (Y. Yung from J. Chun Lab) per well for totoRNAseq analysis
Prepare 5x HiMg RT buffer:[edit]
' | Stock | Final (5x) | Vol for 1 ml |
Tris-HCl pH8.3 | 1M | 250mM | 250ul |
KCl | 1M | 375mM | 375ul |
MgCl2 | 1M | 60mM | 60ul |
dH2O | 315ul | ||
Prepare RT Sort Buffer:[edit]
Component | Volume |
5xRT Buffer | 500ul |
10% Triton-X100 | 10ul |
40U/ul Rnase Inhibitor | 25ul |
ERCC spike-in RNA (1:10^5) | 10ul |
Water | 455ul |
total | 1000ul |
Prepare RNaseIII Sort Buffer:[edit]
Component | Stock | Final (1x) | Volume for 1ml |
Tris-HCl pH8.3 | 1M | 10mM | 10ul |
Triton-X100 | 10% | 0.1% | 10ul |
MgCl2 | 1M | 10mM | 10ul |
NaCl | 2M | 60mM | 30ul |
DTT | 0.1M | 1mM | 10ul |
Rnase Inhibitor | 40U/ul | 1U/ul | 25ul |
ERCC spike-in RNA | 1:10^5 | 10ul | |
Water | 885ul | ||
total | 1000ul | ||
Plate Layout:[edit]
' | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | Sc(1) | Sc(9) | Sc(17) | Sc(25) | Sc(33) | Sc(41) | Sc(49) | Sc(57) | Sc(65) | Sc(73) | Sc(81) | Sc(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) | Sc(24) | Sc(32) | 5cell(40) | 5cell(48) | 10cell(56) | 10cell(64) | Blank(72) | Blank(80) | Blank(88) | Blank(96) |
Sc=Single mouse NPC E12.5 (PI stained and selecting for PI neg); (#)=Indx