Blue:RNA-Seq Experiments:10012013: Difference between revisions
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>RuiLiu |
>B1lake |
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(9 intermediate revisions by 2 users not shown) | |||
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| | | BL_huES8_SingleCell_N2id_89||huES8||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1-24 (no 17)]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id89]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| | ||
|- | |||
| BL_huES8_SingleCell_N2id_90||UHRR||250pg||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id17]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id90]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| | |||
|- | |- | ||
|} | |} | ||
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:''' | :'''Lysis''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 124: | Line 126: | ||
:::''Incubate @ 37C | :::''Incubate @ 37C 10 min'' | ||
:::''Add 1.1ul dH2O | :::''Add 1.1ul dH2O | ||
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::#Add 15ul beads per well | ::#Add 15ul beads per well | ||
::#Allow to bind 15 min | ::#Allow to bind 15 min, combine | ||
::#Remove supernatant and wash 2x with 1ml 80% EtOH | ::#Remove supernatant and wash 2x with 1ml 80% EtOH | ||
::#Air Dry 10min or until cracking first occurs | ::#Air Dry 10min or until cracking first occurs | ||
::#Resuspend beads in | ::#Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul | ||
Line 255: | Line 257: | ||
|2x Buffer||||||||||25ul | |2x Buffer||||||||||25ul | ||
|- | |- | ||
|10uM P1-STRT | |10uM P1-STRT||||||||||1ul | ||
|- | |- | ||
|10uM | |10uM PCR_R_N2_id89,90||||||||||1ul | ||
|- | |- | ||
|H2O||||||||||12ul | |H2O||||||||||12ul | ||
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|98C 15s -> 58C 20s -> 72C 30s||||||||||6x | |98C 15s -> 58C 20s -> 72C 30s||||||||||6x | ||
|- | |- | ||
|98C 10s -> 72C 20s|||||||||| | |98C 10s -> 72C 20s||||||||||11x | ||
|- | |- | ||
|72C, 5min | |72C, 5min | ||
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*Run 5ul on gel: | *Run 5ul on gel: | ||
[[File:10012013_PCR1_gel1.jpg]] | |||
== Final Library Preparation == | == Final Library Preparation == | ||
*Beads purify (0.8x), re-suspend in 10ul | |||
*Run 0.5ul on gel: | |||
[[File:10012013_PCR1_gel2.jpg]] | |||
*PCR of 0.5ul using ILMN_PCR_F/R: | |||
[[File:10012013_PCR2_PCRF-R.jpg]] | |||
*PCR of 0.5ul using ILMN_PCR_F/ERCC130_R227: | |||
[[File:10012013_PCR2_ERCC.jpg]] | |||
Note: | |||
*Pre-PCR TSO control is RnaseIII Smarter TSO control from 09102013 | |||
*To add both 10pg and 250pg UHRR to the plate, lysis buffer (containing ERCC) was removed and 0.4ul UHRR was added - the ERCC expression for id90 should be much smaller than id89 (which has 23 combined wells...) |
Latest revision as of 04:00, 3 October 2013
RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_huES8_SingleCell_N2id_89 | huES8 | Single cell | TSO.r06 | T20V.id1-24 (no 17) | N2.id89 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
BL_huES8_SingleCell_N2id_90 | UHRR | 250pg | TSO.r06 | T20V.id17 | N2.id90 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read |
Plate Layout: Samples and Indexes[edit]
' | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | NC(1) | UHRR 10pg(9) | UHRR 250pg(17) | |||||||||
B | Sc(2) | Sc(10) | Sc(18) | |||||||||
C | Sc(3) | Sc(11) | Sc(19) | |||||||||
D | Sc(4) | Sc(12) | Sc(20) | |||||||||
E | Sc(5) | Sc(13) | Sc(21) | |||||||||
F | Sc(6) | Sc(14) | Sc(22) | |||||||||
G | Sc(7) | Sc(15) | Sc(23) | |||||||||
H | Sc(8) | Sc(16) | 10cells(24) | |||||||||
Note: SC=Single cell; UHRR=Universal Human Reference RNA
Cells Sorted 08122013
Purpose[edit]
- Generate totoRNAseq single cell libraries using modified Smarter TSO (r06) as done in 08202013 that involved r04
- Compare r04 with r06
RNAseIII-Fragmented Library Preparation[edit]
- Cell Sort
Volume RNA 250pg or Single Cells - 1x Lysis Buffer 0.4ul Total 0.4ul
Note: add in UHRR control at this point
- Lysis
Volume RNA 0.4ul 10x RNaseIII Buffer 0.2ul 0.1uM T20V.id 0.1ul dH20 0.55ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 10min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul dH20 0.67ul Total 2ul
- Incubate @ 37C 10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 3.1ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4.1ul 10mM dNTP Mix 1ul 0.1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine (5M) 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min, combine
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul
- TSO
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id89,90 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 11x 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Final Library Preparation[edit]
- Beads purify (0.8x), re-suspend in 10ul
- Run 0.5ul on gel:
- PCR of 0.5ul using ILMN_PCR_F/R:
- PCR of 0.5ul using ILMN_PCR_F/ERCC130_R227:
Note:
- Pre-PCR TSO control is RnaseIII Smarter TSO control from 09102013
- To add both 10pg and 250pg UHRR to the plate, lysis buffer (containing ERCC) was removed and 0.4ul UHRR was added - the ERCC expression for id90 should be much smaller than id89 (which has 23 combined wells...)