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

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(Created page with " == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | al...")
 
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| align="center" style="background:#f0f0f0;"|'''TSO Primer'''
| align="center" style="background:#f0f0f0;"|'''TSO Primer'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers'''
| align="center" style="background:#f0f0f0;"|'''PCR Primers'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2'''
| align="center" style="background:#f0f0f0;"|'''Number of Cycles Amp'''
| align="center" style="background:#f0f0f0;"|'''Type of Seq'''
| align="center" style="background:#f0f0f0;"|'''Type of Seq'''
| align="center" style="background:#f0f0f0;"|'''Reads'''
| align="center" style="background:#f0f0f0;"|'''Reads'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id1||15 Cycles||totoRNAseq||||
| 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_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id2||15 Cycles||totoRNAseq||||
| 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||
|-
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id7-8||18 Cycles||totoRNAseq||||
|}
 
== Plate Layout: Samples and Indexes ==
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#f0f0f0;"|'''6'''
| align="center" style="background:#f0f0f0;"|'''7'''
| align="center" style="background:#f0f0f0;"|'''8'''
| align="center" style="background:#f0f0f0;"|'''9'''
| align="center" style="background:#f0f0f0;"|'''10'''
| align="center" style="background:#f0f0f0;"|'''11'''
| align="center" style="background:#f0f0f0;"|'''12'''
|-
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id9-10||18 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''A'''||NC(1)||UHRR 10pg(9)||UHRR 250pg(17)
|-
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id11-12||21 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''B'''||Sc(2)||Sc(10)||Sc(18)
|-
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id13-14||21 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''C'''||Sc(3)||Sc(11)||Sc(19)
|-
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id3||15 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''D'''||Sc(4)||Sc(12)||Sc(20)
|-
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id4||15 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''E'''||Sc(5)||Sc(13)||Sc(21)
|-
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id5||15 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''F'''||Sc(6)||Sc(14)||Sc(22)
|-
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id6||15 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''G'''||Sc(7)||Sc(15)||Sc(23)
|-
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id15||15 Cycles||totoRNAseq||||
| align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)||Sc(16)||10cells(24)
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id16||15 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id21-22||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id23-24||18 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id25-26||21 Cycles||totoRNAseq||||
|-
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id27-28||21 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id17||15 Cycles||totoRNAseq||||
|-
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id18||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id19||15 Cycles||totoRNAseq||||
|-
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id20||15 Cycles||totoRNAseq||||
|-
|-
|
|}
|}


Note: SC=Single cell; UHRR=Universal Human Reference RNA


[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List]
Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013]
 
UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = RNA from Human Pyramidal Nuclei sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:09232013 09-23-13]; HuLCM = RNA from bulk Human Pyramidal LCM sample ([[File:ERCC bioanalyzer 9.18.13.pdf]])


== 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 ==




:'''RNA PREPARATION'''
:'''Cell Sort'''


::{| {{table}}
::{| {{table}}
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|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|2ng/ul, 200ng/ul, 20ng/ul UHRR||||||||||0.5ul
|RNA 250pg or Single Cells||||||||||-
|-
|1x Lysis Buffer||||||||||0.4ul
|-
|Total||||||||||0.4ul
|-
|}
 
''Note: add in UHRR control at this point''
 
 
 
:'''Lysis'''
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|-
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul
|RNA||||||||||0.4ul
|-
|-
|10x RNaseIII Buffer||||||||||0.2ul
|10x RNaseIII Buffer||||||||||0.2ul
|-
|-
|1uM or 0.1uM (10-100pg) T20V.id1 ||||||||||0.1ul
|0.1uM T20V.id||||||||||0.1ul
|-
|-
|dH20||||||||||0.25ul
|dH20||||||||||0.55ul
|-
|-
|Total||||||||||1.25ul
|Total||||||||||1.25ul
Line 93: Line 106:
:::''Incubate @ 72C 3 min''
:::''Incubate @ 72C 3 min''
:::''Incubate @ 37C 1min''
:::''Incubate @ 37C 1min''
:::''Incubate @ 4C 10min''




Line 112: Line 126:




:::''Incubate @ 37C 5-10 min''
:::''Incubate @ 37C 10 min''


:::''Add 1.1ul dH2O
:::''Add 1.1ul dH2O
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|10mM dNTP Mix||||||||||1ul
|10mM dNTP Mix||||||||||1ul
|-
|-
|1uM or 0.1uM (10-100pg) T20V.ID ||||||||||0.9ul
|0.1uM T20V.ID ||||||||||0.9ul
|-
|-
|}
|}
Line 188: Line 202:


::#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




Line 243: Line 257:
|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
Line 260: Line 274:
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 15x (10pg)
|98C 10s -> 72C 20s||||||||||11x
|-
|-
|72C, 5min
|72C, 5min
Line 270: Line 284:
*Run 5ul on gel:
*Run 5ul on gel:


[[File:09272013_PCR1_gel1.jpg]][[File:09272013_PCR1_gel2.jpg]]
[[File:10012013_PCR1_gel1.jpg]]


Note: U=UHRR; H=Sorted Human Nuclei; L=LCM Human Nuclei; 10=10 minutes RNaseIII fragmentation; 5=5 minutes RNaseIII fragmentation
== Final Library Preparation ==


*Beads purify (0.8x), re-suspend in 10ul
*Run 0.5ul on gel:


[[File:09272013_PCR1_gel3.jpg]][[File:09272013_PCR1_gel4.jpg]]
[[File:10012013_PCR1_gel2.jpg]]


== Final Library Preparation ==
 
*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...)