Blue:RNA-Seq Experiments:10012013: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_huES8_SingleCell_N2id_||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]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id85]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read||
| 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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| align="center" style="background:#f0f0f0;"|'''12'''
| align="center" style="background:#f0f0f0;"|'''12'''
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''A'''||NC(1)||UHRR 10pg(9)||UHRR 250pg(17)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''B'''||Sc(2)||Sc(10)||Sc(18)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''C'''||Sc(3)||Sc(11)||Sc(19)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''D'''||Sc(4)||Sc(12)||Sc(20)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''E'''||Sc(5)||Sc(13)||Sc(21)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''F'''||Sc(6)||Sc(14)||Sc(22)
|-
|-
| align="center" style="background:#f0f0f0;"|'''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)
| align="center" style="background:#f0f0f0;"|'''G'''||Sc(7)||Sc(15)||Sc(23)
|-
|-
| align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)||Sc(16)||10cells(24)||Sc(32)||Sc(40)||10cell(48)||Sc(56)||Sc(64)||10cells(72)||Sc(80)||Sc(88)||10cells(96)
| align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)||Sc(16)||10cells(24)
|-
|-
|  
|  
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Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013]
Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013]


== Purpose ==
== Purpose ==


*Generate totoRNAseq libraries using modified Smarter TSO (r06) and modified r04 TSO to compare efficiencies
*Generate totoRNAseq single cell libraries using modified Smarter TSO (r06) as done in [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08202013 08202013] that involved r04
*Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM
*Compare r04 with r06
*Compare RNAseIII fragmentation times: full (10min) or partial (5min)
 


== RNAseIII-Fragmented Library Preparation ==
== RNAseIII-Fragmented Library Preparation ==
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:'''RNA FRAGMENTATION'''
:'''Lysis'''


::{| {{table}}
::{| {{table}}
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:::''Incubate @ 37C 5-10 min''
:::''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 6ul dH2O, incubate 2min, transfer to new tube - vacuspin 5 min to bring to 3ul
::#Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul




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|2x Buffer||||||||||25ul
|2x Buffer||||||||||25ul
|-
|-
|10uM P1-STRT (Smarter TSO) or PB_PCR_F||||||||||1ul
|10uM P1-STRT||||||||||1ul
|-
|-
|10uM PCR_R_N2_id||||||||||1ul
|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||||||||||12x
|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
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. 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:

File:10012013 PCR1 gel1.jpg

Final Library Preparation[edit]

  • 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...)