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 105: | Line 108: | ||
==Mixed library== | ==Mixed library== | ||
* Mix the 5 library as equal ratio | * Mix the 5 library as equal ratio | ||
* name: | * name: RL_UniRef_totoRNA_Jan16 | ||
* sequencing primer: totoRNAseq Read 1.v2 plus STRT-seq Read 1.v2 | * sequencing primer: totoRNAseq Read 1.v2 plus STRT-seq Read 1.v2 | ||
totoRNAseq Read 1 v2 CCACCGAGATCTACACTCTTTCCCTACACGACG 72 33 | totoRNAseq Read 1 v2 CCACCGAGATCTACACTCTTTCCCTACACGACG 72 33 | ||
STRT-SEQ Read 1 v2 ACCGAGATCTAAGCAGTGGTATCAACGCAGAGT 71.8 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]
- HL141 run [2]