Revision as of 23:42, 25 July 2013 by >B1lake
1.1. Purpose
- Sort single mouse NPCs (Y. Yung from J. Chun Lab) per well for totoRNAseq analysis
5x HiMg RT buffer:
'
|
Stock
|
Final (5x)
|
Vol for 1 ml
|
Tris-HCl pH8.3 |
1M |
250mM |
250ul
|
KCl |
1M |
375mM |
375ul
|
MgCl2 |
1M |
60mM |
60ul
|
dH2O |
|
|
315ul
|
|
RT Sort Buffer:
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
|
|
RNaseIII Sort Buffer:
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:
'
|
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