Rui:LabNotes/SingleCell/2013-1-15: Difference between revisions

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===Procedure===
===Procedure===
* RNA shearing:[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2013-1-14]
* MgCl2 fragmentation:
* RNaseIII digestion:


===Results===
===Results===
Line 52: Line 55:


==DSN treatment==
==DSN treatment==
===Samples===
{| {{table}} border=1
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| 13||38.4||12||460.8||1ng||50ng||1.30||||||||
| 13||38.4||12||460.8||1ng||50ng||1.30||||||||
|}
|}
===Procdure===
* 68C, 2.5 hrs
* DSN for 10min
* minElute in 15ul, 1ul for test
* all for PCR
===Results===
[[File:1.16.13_QPCR.jpg|400px]][[File:1.16.13_gel.jpg|400px]]
==Mixed library==
* Mix the 5 library as equal ratio
* name: RL_UniRef_totoRNA_Jan16
* sequencing primer: totoRNAseq Read 1.v2 plus STRT-seq Read 1.v2
totoRNAseq Read 1 v2 CCACCGAGATCTACACTCTTTCCCTACACGACG 72 33
STRT-SEQ Read 1 v2 ACCGAGATCTAAGCAGTGGTATCAACGCAGAGT 71.8 33
==Data analysis==
* HL141 run [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:TotoRNAseq_on_130120_HL141]

Latest revision as of 18:35, 14 May 2013

totoRNAseq with 10ng/1ng Universal Ref. RNA[edit]

Samples[edit]

' RNA Frag. Purification PNK PAP RT Beads TSO USER PCR_F PCR_R
1 10ng shearing \ yes yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.01
2 SMARTer \ P1-STRT N2.02
3 1ng shearing \ yes yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.03
4 10ng Mg2+ \ yes yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.04
5 SMARTer \ P1-STRT N2.05
6 1ng Mg2+ \ yes yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.06
7 10ng RNaseIII yes \ yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.07
8 SMARTer \ P1-STRT N2.08
9 1ng RNaseIII yes \ yes T20.r01 yes TSO.r02 yes PB_PCR.F N2.09
10 10ng Mg2+ \ yes yes T20.r01 EtOH TSO.r02 yes PB_PCR.F N2.10
11 1ng Mg2+ \ yes yes T20.r01 EtOH TSO.r02 yes PB_PCR.F N2.11
12 10ng Mg2+ \ yes yes T20.r01/TSO.r02 yes PB_PCR.F N2.12
13 1ng Mg2+ \ yes yes T20.r01/TSO.r02 yes PB_PCR.F N2.13

Procedure[edit]

  • RNA shearing:[1]
  • MgCl2 fragmentation:
  • RNaseIII digestion:

Results[edit]

File:1.15.13 QPCR s1-9.jpgFile:1.15.13 gel s1-9.jpg

File:1.15.13 QPCR s10-13.jpgFile:1.15.13 gel s10-13.jpg


DSN treatment[edit]

Samples[edit]

' ng/ul toal ul total ng RNA input for DSN ul total V water 4X hyb total hyb V
1 41.7 12 500.4 ~5ng 40ng 0.96 7.48 0.02 2.5 10.00
2 41.5 12 498 ~5ng 40ng 0.96
3 7.2 12 86.4 1ng 40ng 5.56
4 47.6 12 571.2 ~5ng 40ng 0.84 5.18 2.32 2.5 10
5 43.4 12 520.8 ~5ng 40ng 0.92
6 11.7 12 140.4 1ng 40ng 3.42
7 22.1 12 265.2 ~5ng 50ng 2.26 18.28 6 24.28
8 8.31 12 99.72 ~5ng 50ng 6.02
9 0.767 12 9.204 1ng 7.6ng 10.00
10 40.1 12 481.2 10ng 50ng 1.25 2.65 4.85 2.5 10
11 35.6 12 427.2 1ng 50ng 1.40
12 50.4 12 604.8 10ng 50ng 0.99 2.29 5.21 2.5 10
13 38.4 12 460.8 1ng 50ng 1.30

Procdure[edit]

  • 68C, 2.5 hrs
  • DSN for 10min
  • minElute in 15ul, 1ul for test
  • all for PCR

Results[edit]

File:1.16.13 QPCR.jpgFile:1.16.13 gel.jpg


Mixed library[edit]

  • Mix the 5 library as equal ratio
  • name: RL_UniRef_totoRNA_Jan16
  • sequencing primer: totoRNAseq Read 1.v2 plus STRT-seq Read 1.v2
totoRNAseq Read 1 v2	CCACCGAGATCTACACTCTTTCCCTACACGACG	72	33
STRT-SEQ Read 1 v2	ACCGAGATCTAAGCAGTGGTATCAACGCAGAGT	71.8	33

Data analysis[edit]