Blue:RNA-Seq Experiments:09252013: Difference between revisions
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>B1lake (Created page with "== Purpose == *Sort single mCherry(high)CD184+CD44-CD271- huNSCs (huESC derived) per well for totoRNAseq analysis == Prepare Sort Buffer: == {| {{table}} | align="center"...") |
>B1lake |
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*Sort single mCherry(high)CD184+CD44-CD271- huNSCs (huESC derived) per well for totoRNAseq analysis | *Sort single mCherry(high)CD184+CD44-CD271- huNSCs (huESC derived) per well for totoRNAseq analysis | ||
== Prepare Sort Buffer: == | == Prepare Sort Buffer: == | ||
Line 68: | Line 67: | ||
Sc=Single cell; (#)=Indx | Sc=Single cell; (#)=Indx | ||
'''Notes:''' | |||
*Differentiation involved use of Noggin and TGFbeta inhibitor | |||
*Significant improvement in NSC number was observed | |||
*Sort was done on the BD influx | |||
*Lysis buffer was bubbled just prior to sort | |||
*Given the low volume of lysis buffer there could be well to well variability in volume and as such more variability in ERCC - may want to add ERCC after sort in future... |
Latest revision as of 23:27, 25 September 2013
Purpose[edit]
- Sort single mCherry(high)CD184+CD44-CD271- huNSCs (huESC derived) per well for totoRNAseq analysis
Prepare Sort Buffer:[edit]
Component | Stock | Final (1x) | Volume |
dH2O | 183ul | ||
Triton-X100 | 10% | 0.1% | 2ul |
Rnase Inhibitor | 40U/ul | 1U/ul | 5ul |
ERCC spike-in RNA | 1:10^5 | 10ul | |
total | 200ul | ||
- Note: using 0.4ul per well and generating bubbles just prior to sort
Plate Layout:[edit]
' | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 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) |
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:
- Differentiation involved use of Noggin and TGFbeta inhibitor
- Significant improvement in NSC number was observed
- Sort was done on the BD influx
- Lysis buffer was bubbled just prior to sort
- Given the low volume of lysis buffer there could be well to well variability in volume and as such more variability in ERCC - may want to add ERCC after sort in future...