Blue:RNA-Seq Experiments:06182013

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1.1. Purpose[edit]

  • Sort single Pyramidal neuronal nuclei per well for totoRNAseq analysis


5x HiMg RT buffer:[edit]

Component Stock Final (5x) Vol for 1 ml
Tris-HCl pH8.3 1M 250mM 250ul
KCl 1M 375mM 375ul
MgCl2 1M 60mM 60ul
dH2O 315ul

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

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 Cell Nucleus; (#)=Indx

Nuclei Preparation: Yun Yung and Gwen Kaeser from J. Chun lab. Labeled 1230 non-diseased. Sorted based on size - larger size for pyramidal neurons

Pool Sample: Sorted --75x105 nuclei for pooled RNA analysis (Note: accidentally left in centrifuge for a few hours...)


Sort Parameters:

File:Pyramidal.jpg

1.2. Purpose[edit]

  • Sort single B6 mESCs, 2 plates sorted into above buffer


1. SF = standard plate format indicated above

2. LF = Lane format of:

A - 1 Cells

B - 1 Cells

C - 1 Cells

D - 1 Cells

E - 5 Cells

F - 5 Cells

G - 10 Cells

H - 10 Cells