Blue:RNA-Seq Experiments:03052014: Difference between revisions
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| ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min ||TSO.r06||T20VN_id11||N2.id12||totoRNAseq | | ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min ||TSO.r06||T20VN_id11||N2.id12||totoRNAseq | ||
|- | |||
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||T20VN_id11||N2.id13-14||totoRNAseq | |||
|- | |||
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||T20VN_id11||N2.id15-16||totoRNAseq | |||
|- | |||
| ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||C1-P1-RNA-TSO||T20VN_id1-10||N2.id17||totoRNAseq | |||
|- | |||
| ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min ||C1-P1-RNA-TSO||T20VN_id11||N2.id18||totoRNAseq | |||
|- | |- | ||
| ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq | | ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq | ||
Line 216: | Line 224: | ||
:'''Beads Purification''' | :'''Beads Purification''' | ||
::#Add | ::#Add 25ul beads per well | ||
::#Allow to bind 15 min | ::#Allow to bind 15 min | ||
::#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 | ::#Resuspend beads in 6ul dH20; incubate 2min RT; transfer to new tube and vacuum spin to ~3ul (~8min) | ||
== cDNA Preparation: Mg-based Fragmentation == | == cDNA Preparation: Mg-based Fragmentation == | ||
Line 406: | Line 414: | ||
*Run 5ul on gel: | *Run 5ul on gel: | ||
[[File:03052014_PCR1_Gel1_toto.jpg| | [[File:03052014_PCR1_Gel1_toto.jpg|300px]] | ||
Line 413: | Line 421: | ||
* Beads purified (0.8x) twice and eluted in 10ul. | * Beads purified (0.8x) twice and eluted in 10ul. | ||
'''Repeat RNaseIII Protocol using Sten TSO''' | |||
:*Repeated exactly the same way except with the following substitutions: | |||
:**TSO.r06 replaced with C1-P1-RNA-TSO | |||
:**P1-STRT replaced with C1-P1-PCR-2 | |||
:*Run 5ul on gel: | |||
[[File:03052014_PCR1_Gel1_toto_sten.jpg|200px]] | |||
Note: Possible mistake with the quantification of the input RNA obtained from Illumina - may be double that intended. Indicates that the efficiency of this TSO in the totoRNAseq protocol might be too low to be truly viable... | |||
== UMI-STRT-Seq Library Preparation == | == UMI-STRT-Seq Library Preparation == | ||
Line 641: | Line 662: | ||
Results: Appears that the Smart-seq2 protocol is also incompatible with the Mg RNA fragmentation protocol (likely due to the enzymatics PAP). For RNaseIII fragmented RNA, the efficiency looks to be too low to amplify 10pg libraries... therefore the Smart-seq2 adaptation | Results: Appears that the Smart-seq2 protocol is also incompatible with the Mg RNA fragmentation protocol (likely due to the enzymatics PAP). For RNaseIII fragmented RNA, the efficiency looks to be too low to amplify 10pg libraries... therefore the Smart-seq2 adaptation may not work well enough to continue. cDNA quantification is needed to determine which samples can be used for Nextera library preparation. | ||
== Quartz-Seq Library Preparation == | == Quartz-Seq Library Preparation == | ||
Line 765: | Line 786: | ||
[[File:03052014_PCR1_Gel1_Quartz.jpg|500px]] | [[File:03052014_PCR1_Gel1_Quartz.jpg|500px]] | ||
Results: Mg protocol also is problematic for Quartz-seq. I'm not sure why this is the case since no RNA adaptor primer was used... Looks like the RNaseIII protocol did work down to the 10pg level, though quantification is needed to determine whether there is enough yield for Nextera library preparation. Note that while the yields look lower, less sample was run on the gel compared to totoRNAseq (5ul of 107ul vs 5ul of 50ul). | |||
== MARS-Seq Library Preparation == | == MARS-Seq Library Preparation == | ||
Line 1,023: | Line 1,046: | ||
[[File:03052014_MARS_PCR1.jpg|400px]] | [[File:03052014_MARS_PCR1.jpg|400px]] | ||
== Nextera | Results: Amplification pattern implies problem with protocol... needs further optimization... | ||
== Final Library Preparation == | |||
''cDNA Quantification'' | |||
:'''Quantification of 1ul out of 10ul Beads purified libraries using Qubit:''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Condition''' | |||
| align="center" style="background:#f0f0f0;"|'''[cDNA] ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''Final Library''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume to Combine''' | |||
| align="center" style="background:#f0f0f0;"|'''N2 Barcode''' | |||
| align="center" style="background:#f0f0f0;"|'''NexteraXT''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 0.1-0.2ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume dH2O''' | |||
| align="center" style="background:#f0f0f0;"|'''Nextera Barcode''' | |||
| align="center" style="background:#f0f0f0;"|'''Sten Tagmentation''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 1ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume dH20''' | |||
| align="center" style="background:#f0f0f0;"|'''Sten Barcode''' | |||
|- | |||
| 1||TotoRNAseq Mg 1ng||8.95||Yes||2||1||N/A||||||||N/A|||||| | |||
|- | |||
| 2||TotoRNAseq Mg 1ng||8.84||Yes||2.02||2||N/A||||||||N/A|||||| | |||
|- | |||
| 3||TotoRNAseq Mg 100pg||8.57||Yes||2.09||3||N/A||||||||N/A|||||| | |||
|- | |||
| 4||TotoRNAseq Mg 100pg||10||Yes||1.79||4||N/A||||||||N/A|||||| | |||
|- | |||
| 5||TotoRNAseq Mg 10x10pg||15.2||Yes||N/A||5||N/A||||||||N/A|||||| | |||
|- | |||
| 6||TotoRNAseq Mg NTC||4.04||Yes||2||6||N/A||||||||N/A|||||| | |||
|- | |||
| 7||TotoRNAseq RIII 1ng||3.13||Yes||1||7||N/A||||||||N/A|||||| | |||
|- | |||
| 8||TotoRNAseq RIII 1ng||2.96||Yes||1.06||8||N/A||||||||N/A|||||| | |||
|- | |||
| 9||TotoRNAseq RIII 100pg||1.38||Yes||2.27||9||N/A||||||||N/A|||||| | |||
|- | |||
| 10||TotoRNAseq RIII 100pg||0.977||Yes||3.20||10||N/A||||||||N/A|||||| | |||
|- | |||
| 11||TotoRNAseq RIII 10x10pg||1.39||Yes||N/A||11||N/A||||||||N/A|||||| | |||
|- | |||
| 12||TotoRNAseq RIII NTC||-||Yes||2||12||N/A||||||||N/A|||||| | |||
|- | |||
| 13||TotoRNAseq RIII StenTSO 1ng||1.21||Yes||1||13||N/A||||||||N/A|||||| | |||
|- | |||
| 14||TotoRNAseq RIII StenTSO 1ng||1.54||Yes||0.785714286||14||N/A||||||||N/A|||||| | |||
|- | |||
| 15||TotoRNAseq RIII StenTSO 100pg||0.948||Yes||1.276371308||15||N/A||||||||N/A|||||| | |||
|- | |||
| 16||TotoRNAseq RIII StenTSO 100pg||0.844||Yes||1.433649289||16||N/A||||||||N/A|||||| | |||
|- | |||
| 17||TotoRNAseq RIII StenTSO 10x10pg||0.19||Yes||6.368421053||17||N/A||||||||N/A|||||| | |||
|- | |||
| 18||TotoRNAseq RIII StenTSO NTC||-||Yes||1||18||N/A||||||||N/A|||||| | |||
|- | |||
| 19||STRT RIII 1ng||3.54||No||||||N/A||||||||Yes||1.694915254||4.305084746||C1-TN5-1 | |||
|- | |||
| 20||STRT RIII 1ng||3.35||No||||||N/A||||||||Yes||1.791044776||4.208955224||C1-TN5-2 | |||
|- | |||
| 21||STRT RIII 100pg||2.78||No||||||N/A||||||||Yes||2.158273381||3.841726619||C1-TN5-3 | |||
|- | |||
| 22||STRT RIII 100pg||2.99||No||||||N/A||||||||Yes||2.006688963||3.993311037||C1-TN5-4 | |||
|- | |||
| 23||STRT RIII 10pg||2.42||No||||||N/A||||||||Yes||2.479338843||3.520661157||C1-TN5-5 | |||
|- | |||
| 24||STRT RIII 10pg||2.22||No||||||N/A||||||||Yes||2.702702703||3.297297297||C1-TN5-6 | |||
|- | |||
| 25||STRT RIII 10pg||2.86||No||||||N/A||||||||Yes||2.097902098||3.902097902||C1-TN5-7 | |||
|- | |||
| 26||STRT RIII NTC||0.696||No||||||N/A||||||||Yes||6||0||C1-TN5-8 | |||
|- | |||
| 27||Smart2 RIII 1ng||1.48||No||||||Yes||0.68||4.32||S501/N701||N/A|||||| | |||
|- | |||
| 28||Smart2 RIII 1ng||1.47||No||||||Yes||0.68||4.32||S502/N702||N/A|||||| | |||
|- | |||
| 29||Smart2 RIII 100pg||0.52||No||||||Yes||1.92||3.08||S503/N703||N/A|||||| | |||
|- | |||
| 30||Smart2 RIII 100pg||0.49||No||||||Yes||2.04||2.96||S504/N704||N/A|||||| | |||
|- | |||
| 31||Smart2 RIII 10pg||0.13||No||||||Yes||3.85||1.15||S505/N705||N/A|||||| | |||
|- | |||
| 32||Smart2 RIII 10pg||0.12||No||||||Yes||4.17||0.83||S506/N706||N/A|||||| | |||
|- | |||
| 33||Smart2 RIII 10pg||-||No||||||Yes||5.00||0.00||S507/N707||N/A|||||| | |||
|- | |||
| 34||Smart2 RIII NTC||-||No||||||Yes||5.00||0.00||S508/N708||N/A|||||| | |||
|- | |||
| 35||Quartz RIII 1ng||13.8||No||||||Yes||0.58||39.42||S501/N709||N/A|||||| | |||
|- | |||
| 36||Quartz RIII 1ng||10.4||No||||||Yes||0.77||39.23||S502/N710||N/A|||||| | |||
|- | |||
| 37||Quartz RIII 100pg||3.49||No||||||Yes||0.86||14.14||S503/N711||N/A|||||| | |||
|- | |||
| 38||Quartz RIII 100pg||3.73||No||||||Yes||0.80||14.20||S504/N712||N/A|||||| | |||
|- | |||
| 39||Quartz RIII 10pg||1.95||No||||||Yes||1.54||13.46||S505/N701||N/A|||||| | |||
|- | |||
| 40||Quartz RIII 10pg||3.63||No||||||Yes||0.83||14.17||S506/N702||N/A|||||| | |||
|- | |||
| 41||Quartz RIII 10pg||4.93||No||||||Yes||0.61||14.39||S507/N703||N/A|||||| | |||
|- | |||
| 42||Quartz RIII NTC||1.83||No||||||Yes||1.64||13.36||S508/N704||N/A|||||| | |||
|} | |||
''Nextera Tagmentation'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagment DNA Buffer||||||||||5ul | |||
|- | |||
|Amplification Tagment Mix||||||||||2.5ul | |||
|- | |||
|Diluted Sample||||||||||2.5ul | |||
|- | |||
|Total||||||||||10ul | |||
|- | |||
|} | |||
::*Vortex and spin | |||
::*Incubate 55C 5 min | |||
::*Hold 10C | |||
::*Once sample reaches 10C add 2.5ul of NT buffer in block | |||
::*Vortex and spin | |||
''Nextera Amplification'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagmented Sample||||||||||12.5ul | |||
|- | |||
|Nextera PCR Mix||||||||||7.5ul | |||
|- | |||
|S5xx Primer||||||||||2.5ul | |||
|- | |||
|S7xx Primer||||||||||2.5ul | |||
|- | |||
|Total||||||||||20ul | |||
|- | |||
|} | |||
::''Mix and centrifuge'' | |||
::{| {{table}} | |||
|- | |||
|72C 3 min|||||||||| | |||
|- | |||
|95C 30s|||||||||| | |||
|- | |||
|95C 10s -> 55C 30s -> 72C 60s||||||||||12x | |||
|- | |||
|72C, 5min | |||
|- | |||
|} | |||
::*Run 2.5ul on gel: | |||
[[File:03052014_PCR2_Gel1_Nextera.jpg|500px]] | |||
::*Combined 5ul of each for Smart2 | |||
::*Combined 5ul each of 1ng/100pg/NTC and 7.5ul each of 10pg for Quartz-seq | |||
::*Beads purified twice (0.8x volume) and resuspended in 10ul | |||
''Sten Tagmentation'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|5x Tagmentation Buffer||||||||||5ul | |||
|- | |||
|Transposome stock (Epicenter)||||||||||6ul | |||
|- | |||
|Sample||||||||||3ul | |||
|- | |||
|dH2O||||||||||11ul | |||
|- | |||
|Total||||||||||25ul | |||
|- | |||
|} | |||
::*Incubate @55C for 8 min | |||
::*Cool on ice | |||
::*Wash 100ul Dynabeads MyOne Streptavidin C1 Beads with 2xBWT and resuspended in 2ml 2xBWT | |||
::*Add 20ul beads to each well and incubate 5min | |||
::*Combine beads, immobilize and discard supernatant | |||
::*Wash beads with 100ul TNT | |||
::*Wash beads with 100ul Qiagen Qiaquick PB | |||
::*Wash beads twice with 100ul TNT | |||
::*Resuspend beads in 100ul restriction mix: | |||
:::*10ul NEBuffer 4 | |||
:::*2ul PvuI-HF (0.4U/ul) | |||
:::*88ul dH2O | |||
::*Incubate at 37C for 1 hour | |||
::*Wash beads 3x with TNT | |||
::*Elute in 10ul 100mM NaOH, incubate 5 min then remove to new tube | |||
::*Neutralize by adding: | |||
:::*10ul 100mM HCl | |||
:::*5ul Neutralization buffer | |||
::*Check library size by gel (ran 5ul on UREA gel): | |||
[[File:03052014_PCR2_Gel1_STRT.jpg|100px]] | |||
::Ran Sybr PCR using C1_P1_PCR_2/C1_TN5_U for 12 cycles (25ul total) and ran 5ul on gel: | |||
[[File:03052014_PCR2_Gel2_STRT.jpg|100px]] | |||
::[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:Data/2014-5-12 Final Libraries Ran on Gel] |
Latest revision as of 01:13, 19 June 2014
Experimental Overview[edit]
File:TotoRNAseq expanded optimization3.jpg
Purpose[edit]
- Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
- Comparison of Mg-based and RNaseIII based fragmentation methods
RNA-Seq Library Information[edit]
Library ID | Sample | Condition | 5' Adaptor | 3' Adaptor | N2 PCR Rev Primer | RNA-Seq Protocol |
BL_totoRNAseq_1ngUHRR_ | 2x1ng UHRR | 2min Mg Frag/Enzymatics PAP 5 min | TSO.r06 | T20VN_id1 | N2.id1-2 | totoRNAseq |
2x100pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | TSO.r06 | T20VN_id1 | N2.id3-4 | totoRNAseq | |
10x10pg UHRR (combined) | 2min Mg Frag/Enzymatics PAP 5 min | TSO.r06 | T20VN_id1-10 | N2.id5 | totoRNAseq | |
NTC | 2min Mg Frag/Enzymatics PAP 5 min | TSO.r06 | T20VN_id11 | N2.id6 | totoRNAseq | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | TSO.r06 | T20VN_id1 | N2.id7-8 | totoRNAseq | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | TSO.r06 | T20VN_id1 | N2.id9-10 | totoRNAseq | |
10x10pg UHRR (combined) | 0.05U RNase3 Frag/Affymetrix PAP 4 min | TSO.r06 | T20VN_id1-10 | N2.id11 | totoRNAseq | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | TSO.r06 | T20VN_id11 | N2.id12 | totoRNAseq | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | C1-P1-RNA-TSO | T20VN_id11 | N2.id13-14 | totoRNAseq | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | C1-P1-RNA-TSO | T20VN_id11 | N2.id15-16 | totoRNAseq | |
10x10pg UHRR (combined) | 0.05U RNase3 Frag/Affymetrix PAP 4 min | C1-P1-RNA-TSO | T20VN_id1-10 | N2.id17 | totoRNAseq | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | C1-P1-RNA-TSO | T20VN_id11 | N2.id18 | totoRNAseq | |
2x1ng UHRR | 2min Mg Frag/Enzymatics PAP 5 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
2x100pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
3x10pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
NTC | 2min Mg Frag/Enzymatics PAP 5 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
3x10pg UHRR | 0.05U RNase3 Frag/Affymetrix PAP 4 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | C1-P1-RNA-TSO | C1-P1-T31 | C1-TN5-x | totoRNAseq/UMI-STRT-Seq | |
2x1ng UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
2x100pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
3x10pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
NTC | 2min Mg Frag/Enzymatics PAP 5 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
3x10pg UHRR | 0.05U RNase3 Frag/Affymetrix PAP 4 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | Smart2_TSO | Smart2_T30VN | N7xx/N5xx | totoRNAseq/Smart-Seq2 | |
2x1ng UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x100pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
3x10pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
NTC | 2min Mg Frag/Enzymatics PAP 5 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
3x10pg UHRR | 0.05U RNase3 Frag/Affymetrix PAP 5 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Lig_NNNX4_id01-2 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq | |
2x100pg UHRR | 2min Mg Frag/Enzymatics PAP 5 min | Lig_NNNX4_id03-4 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq | |
10x10pg UHRR (combined) | 2min Mg Frag/Enzymatics PAP 5 min | Lig_NNNX4_id05 | MARS_T20N_id01-10 | n/a | totoRNAseq/MARS-Seq | |
NTC | 2min Mg Frag/Enzymatics PAP 5 min | Lig_NNNX4_id06 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq | |
2x1ng UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Lig_NNNX4_id01-2 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq | |
2x100pg UHRR | 0.1U RNase3 Frag/Affymetrix PAP 10 min | Lig_NNNX4_id3-4 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq | |
10x10pg UHRR (combined) | 0.05U RNase3 Frag/Affymetrix PAP 4 min | Lig_NNNX4_id5 | MARS_T20N_id01-10 | n/a | totoRNAseq/MARS-Seq | |
NTC | 0.05U RNase3 Frag/Affymetrix PAP 4 min | Lig_NNNX4_id6 | MARS_T20N_id01 | n/a | totoRNAseq/MARS-Seq |
Primer sequences [1]
cDNA Preparation: RNAseIII-Based Fragmentation[edit]
- RNA
Volume 2ng/ul or 0.2ng/ul or 0.02ng/ul 0.5ul 1:10E4 or 1:10E5 or 1:10E6 ERCC 0.2ul xuM PolyT primer (see below) 0.1ul dH2O 0.2ul Total 1ul
Note: NTC with 1:10E6 ERCC and no RNA - treated as 10pg...
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 1 min
- 3' Tailing
Volume fRNA 2ul 5x RT buffer 0.5ul 1mM ATP 0.5ul 600U/ul Affymetrix PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.6ul Total 4ul
- Incubate @ 37C 4-10 min
- Add 1ul of 0.1uM T20VN.id [totoRNAseq]
- Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]
- Add 1ul of 10uM Smart2-T30VN [Smart-seq2]
- Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]
- Add 1ul of 2uM MARS_T20N_id [MARS-Seq]
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1.5ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul Total 10ul
- Incubate @ 42C 60 min
- For MARS-seq - continue to that section
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH20; incubate 2min RT; transfer to new tube and vacuum spin to ~3ul (~8min)
cDNA Preparation: Mg-based Fragmentation[edit]
- RNA FRAGMENTATION
Volume 2ng/ul or 0.2ng/ul or 0.02ng/ul 0.5ul 1:10E4 or 1:10E5 or 1:10E6 ERCC 0.2ul 5x RT buffer 1ul dH2O 0.3ul Total 2ul
- Incubate @ 94C 2 min
- Transfer to ice
- End Repair
Volume fRNA 2ul 1mM ATP 0.4ul 100mM DTT 0.1ul RNase Inhibitor 0.1ul PNK 0.5ul Total 3.1ul
- Incubate @ 37C 30 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 1mM ATP 0.5ul 0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul Total 4.1
- Incubate @ 37C 5 min
- Transfer to Ice
- Add 1ul of 0.1uM T20VN.id [totoRNAseq]
- Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]
- Add 1ul of 10uM Smart2-T30VN [Smart-seq2]
- Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]
- Add 1ul of 2uM MARS_T20N_id [MARS-Seq]
- Reverse Transcription
Volume PAP RNA 5.1ul 5xRT Buffer 1ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul dH2O 0.4ul Total 10ul
- Incubate @ 42C 60 min
- For MARS-seq - continue to that section
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul
totoRNASeq Library Preparation[edit]
- TSO Reaction
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 20min
- Incubate @70C 10min
- PCR (PhusionHF)
Volume TSO Reaction 10ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (1ng); 12x (100pg) 72C, 5min
- Run 5ul on gel:
File:03052014 PCR1 Gel1 toto.jpg
Results: Decent amplification for all conditions. Negative control (NC) which does have ERCC (10pg level) shows a distinct band at around 300bp for both Mg protocol (above) and the RNaseIII protocol (see STRT gel below). Mg protocol does show more high molecular weight fragments.
- Beads purified (0.8x) twice and eluted in 10ul.
Repeat RNaseIII Protocol using Sten TSO
- Repeated exactly the same way except with the following substitutions:
- TSO.r06 replaced with C1-P1-RNA-TSO
- P1-STRT replaced with C1-P1-PCR-2
- Run 5ul on gel:
- Repeated exactly the same way except with the following substitutions:
File:03052014 PCR1 Gel1 toto sten.jpg
Note: Possible mistake with the quantification of the input RNA obtained from Illumina - may be double that intended. Indicates that the efficiency of this TSO in the totoRNAseq protocol might be too low to be truly viable...
UMI-STRT-Seq Library Preparation[edit]
- TSO Reaction
Volume Purified cDNA 3ul 5x First Strand buffer 1.4ul 10mM dNTP mix 0.7ul 100mM DTT 0.175ul 10uM C1-P1-RNA-TSO 0.7ul RNase Inhibitor 0.175ul SuperScriptII 0.7ul dH2O 0.15ul Total 7ul
- Incubate @42C 20min
- Incubate @70C 10min
- PCR (Advantage)
Volume TSO Reaction 7ul 10x Advantage 2 Buffer 7ul 10uM C1-P1-PCR-2 2.8ul dNTPs 2.8ul Advantage 2 Polymerase 2.8ul H2O 47.6ul Total 70ul
95C 60 sec 95C 20s -> 58C 4min -> 68C 6min 5x 95C 20s -> 64C 30s -> 68C 1min 10x (1ng); 13x (100pg); 16x (10pg) 72C, 10min
- Run 7ul on gel:
File:03052014 PCR1 Gel1 STRT.jpg
Results: Mg protocol does not seem to be compatible with Sten's latest STRT protocol (using RNA TSO) - possible carry over of Enzymatics PAP through beads purification. RNaseIII protocol (Affymetrics PAP) seems fine for all RNA amounts, but does show a ~600bp band. The NC for the RNaseIII STRT protocol does seem clean. Amplification levels for the RNaseIII protocol are lower than the totoRNAseq protocol.
- Beads Purified (0.9x) twice and eluted in 10ul. Quantification using Qubit:
Smart-Seq2 Library Preparation[edit]
- TSO Reaction
Volume Purified cDNA 3ul 5x First Strand buffer 2ul 10mM dNTP mix 1ul 100mM DTT 0.5ul 100uM Smart2-TSO 0.1ul MgCl2 (1M) 0.06ul Betaine (5M) 2ul RNase Inhibitor 0.25ul SuperScriptII 0.5ul dH2O 0.59ul Total 10ul
- Incubate @42C 20min
- Incubate @70C 10min
- Incubate @10C hold
- PCR (KAPA-HiFi)
Volume TSO Reaction 10ul 2x KAPA HiFi Hotstart ReadyMix 12.5ul IS PCR Primer (10uM) 0.25ul H2O 2.25ul Total 25ul
98C 3min 98C 20s -> 67C 15s -> 72C 1min 15x (1ng); 18x (100pg); 21x (10pg) 72C, 5min
File:03052014 PCR1 Gel1 Smart2.jpg
Results: Can see that the PCR did not work. Maybe the conditions are sub-optimal for fragmented RNA. Could try similar conditions as the STRT-seq or totoRNAseq...
Repeat Experiment
- TSO Reaction
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 20min
- Incubate @70C 10min
- PCR (PhusionHF)
Volume TSO Reaction 10ul 2x Buffer 25ul IS PCR Primer (10uM) 2ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 64C 20s -> 72C 20s 9x (1ng); 12x (100pg); 15x (10pg) 72C, 5min
- Run 5ul on gel:
File:03052014 PCR1 Gel1 Smart2 2.jpg
Results: Appears that the Smart-seq2 protocol is also incompatible with the Mg RNA fragmentation protocol (likely due to the enzymatics PAP). For RNaseIII fragmented RNA, the efficiency looks to be too low to amplify 10pg libraries... therefore the Smart-seq2 adaptation may not work well enough to continue. cDNA quantification is needed to determine which samples can be used for Nextera library preparation.
Quartz-Seq Library Preparation[edit]
RT Primer Removal
Volume Purified cDNA 3ul 10x PCR Buffer 0.4ul 10x Exo I buffer 0.2ul 100mM DTT 0.2ul Exonuclease I 0.6ul dH2O 1.6ul Total 6ul
Note: can add directly to beads and transfer to new tube
- Incubate @37C 30min
- Incubate @80C 20min
- Incubate @4C
Poly-A Tailing
Volume ExoI RXN 6ul 10x PCR Buffer 0.5ul 100mM dATP 0.15ul RNaseH 0.12ul TdT enzyme 0.42ul dH2O 3.81ul Total 11ul
- Incubate @37C 50 sec
- Incubate @65C 10min
- Incubate @4C
Second Strand Synthesis
Volume Poly-A Tailed Sample 11ul 2x Terra Direct PCR Buffer 25ul 10uM Tagging Primer 0.32ul Terra Polymerase 2ul dH2O 18.68ul Total 57ul
- Incubate @98C 2min 10 sec
- Incubate @40C 1min
- Incubate @68C 5min
- Transfer to ice
PCR Amplification
Volume 2nd Strand Sample 57ul 2x Terra Direct PCR Buffer 25ul 100uM PCR Primer 1ul dH2O 24ul Total 107ul
68C 1 sec 98C 10s -> 65C 15s -> 68C 5min 15x (1ng); 18x (100pg); 21x (10pg) 68C, 5min
Ran 5ul onto gel:
File:03052014 PCR1 Gel1 Quartz.jpg
Results: Mg protocol also is problematic for Quartz-seq. I'm not sure why this is the case since no RNA adaptor primer was used... Looks like the RNaseIII protocol did work down to the 10pg level, though quantification is needed to determine whether there is enough yield for Nextera library preparation. Note that while the yields look lower, less sample was run on the gel compared to totoRNAseq (5ul of 107ul vs 5ul of 50ul).
MARS-Seq Library Preparation[edit]
Exonuclease Digestion
- Add 1ul Exonuclease I (1:10 diluted in 1x exonuclease buffer)to each RT reaction
- Incubate at 37C for 30min
- Incubate at 80C for 10min
- Beads Purification:
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 17ul dH2O, incubate 2min, transfer to new tube
(Note: stored overnight at -20C at this point)
Second Strand Synthesis
Volume dH20 17ul 10x NEB second strand synthesis buffer 2ul Second strand synthesis enzyme 1ul Total 20ul
- Incubate @ 16C for 2.5 hours
IVT
- Add 28ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in:
Volume 10x Reaction Buffer (T7 high yield NEB) 2ul UTP 2ul ATP 2ul CTP 2ul GTP 2ul T7 polymerase 2ul dH2O 8ul Total 20ul
- Incubate 16 hours (overnight) @ 37C'
- Add 1ul Turbo DNase I and incubate @ 37C 15min
- Beads Purification:
- Add 24ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH20, transfer to new tube, vacuspin to 3ul
Adaptor Ligation
Volume aRNA 3ul 100uM Lig_NNNX4_idx 1ul
- Incubate @ 70C for 3min
Volume aRNA/adaptor 4ul 10x T4 Ligase buffer 1ul DMSO 0.95ul 10mM ATP 1ul PEG8000 2ul T4 RNA Ligase I(10U/ul) 1ul dH2O 0ul Total 10ul
- Incubate at 22C for 2hrs
Quantification:
- Removed 4ul for RT (see below)
- Added 14ul to 6ul ligated RNA
- Beads purified (1.2x)
- Quantified by Qubit:
Invitrogen ' ' ' ' ' ' Qubit(TM) Fluorometer 3/27/2014 2:26:11 PM Quant-iT RNA Sample Concentration in the Qubit µL used Dilution Sample Concentration RIII_id1 79.3 ng/mL 1 200.0 15.9 µg/mL RIII_id2 115 ng/mL 1 200.0 23.0 µg/mL RIII_id3 46.1 ng/mL 1 200.0 9.21 µg/mL RIII_id4 46.5 ng/mL 1 200.0 9.30 µg/mL RIII_id5 Out Of Range (too low) RIII_id6 (NTC) 102 ng/mL 1 200.0 20.4 µg/mL
Reverse Transcription
Volume Ligated RNA 4ul 10uM MARS_RT_2 0.5ul 10mM dNTP 0.5ul Total 5ul
- Incubate @ 65C 5min
- Incubate on ice 1min
Volume RNA/Primer 5ul 10x RT Buffer 1ul 25mM MgCl2 0.4ul 100mM DTT 1ul RNase Inhibitor (40U/ul) 0.5ul SSIII (200U/ul) 0.5ul dH2O 1.6ul Total 10ul
- Incubate @42C 10min
- Incubate @50C 50min
- Incubate @85C 5min
Beads Purify
- Add 30ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH20, transfer to new tube
PCR Amplification
Volume cDNA 10ul SybrGreen KAPA 2x MM 25ul 10uM P5_Rd1_PCR_F 2.5ul 10uM P7_Rd2_PCR_R 2.5ul dH2O 10ul
95C 30 sec 95C 15s -> 60C 20s -> 72C 30s up to 20x 72C, 10min
Results: Amplification pattern implies problem with protocol... needs further optimization...
Final Library Preparation[edit]
cDNA Quantification
- Quantification of 1ul out of 10ul Beads purified libraries using Qubit:
Sample | Condition | [cDNA] ng/ul | Final Library | Volume to Combine | N2 Barcode | NexteraXT | Volume for 0.1-0.2ng/ul | Volume dH2O | Nextera Barcode | Sten Tagmentation | Volume for 1ng/ul | Volume dH20 | Sten Barcode |
1 | TotoRNAseq Mg 1ng | 8.95 | Yes | 2 | 1 | N/A | N/A | ||||||
2 | TotoRNAseq Mg 1ng | 8.84 | Yes | 2.02 | 2 | N/A | N/A | ||||||
3 | TotoRNAseq Mg 100pg | 8.57 | Yes | 2.09 | 3 | N/A | N/A | ||||||
4 | TotoRNAseq Mg 100pg | 10 | Yes | 1.79 | 4 | N/A | N/A | ||||||
5 | TotoRNAseq Mg 10x10pg | 15.2 | Yes | N/A | 5 | N/A | N/A | ||||||
6 | TotoRNAseq Mg NTC | 4.04 | Yes | 2 | 6 | N/A | N/A | ||||||
7 | TotoRNAseq RIII 1ng | 3.13 | Yes | 1 | 7 | N/A | N/A | ||||||
8 | TotoRNAseq RIII 1ng | 2.96 | Yes | 1.06 | 8 | N/A | N/A | ||||||
9 | TotoRNAseq RIII 100pg | 1.38 | Yes | 2.27 | 9 | N/A | N/A | ||||||
10 | TotoRNAseq RIII 100pg | 0.977 | Yes | 3.20 | 10 | N/A | N/A | ||||||
11 | TotoRNAseq RIII 10x10pg | 1.39 | Yes | N/A | 11 | N/A | N/A | ||||||
12 | TotoRNAseq RIII NTC | - | Yes | 2 | 12 | N/A | N/A | ||||||
13 | TotoRNAseq RIII StenTSO 1ng | 1.21 | Yes | 1 | 13 | N/A | N/A | ||||||
14 | TotoRNAseq RIII StenTSO 1ng | 1.54 | Yes | 0.785714286 | 14 | N/A | N/A | ||||||
15 | TotoRNAseq RIII StenTSO 100pg | 0.948 | Yes | 1.276371308 | 15 | N/A | N/A | ||||||
16 | TotoRNAseq RIII StenTSO 100pg | 0.844 | Yes | 1.433649289 | 16 | N/A | N/A | ||||||
17 | TotoRNAseq RIII StenTSO 10x10pg | 0.19 | Yes | 6.368421053 | 17 | N/A | N/A | ||||||
18 | TotoRNAseq RIII StenTSO NTC | - | Yes | 1 | 18 | N/A | N/A | ||||||
19 | STRT RIII 1ng | 3.54 | No | N/A | Yes | 1.694915254 | 4.305084746 | C1-TN5-1 | |||||
20 | STRT RIII 1ng | 3.35 | No | N/A | Yes | 1.791044776 | 4.208955224 | C1-TN5-2 | |||||
21 | STRT RIII 100pg | 2.78 | No | N/A | Yes | 2.158273381 | 3.841726619 | C1-TN5-3 | |||||
22 | STRT RIII 100pg | 2.99 | No | N/A | Yes | 2.006688963 | 3.993311037 | C1-TN5-4 | |||||
23 | STRT RIII 10pg | 2.42 | No | N/A | Yes | 2.479338843 | 3.520661157 | C1-TN5-5 | |||||
24 | STRT RIII 10pg | 2.22 | No | N/A | Yes | 2.702702703 | 3.297297297 | C1-TN5-6 | |||||
25 | STRT RIII 10pg | 2.86 | No | N/A | Yes | 2.097902098 | 3.902097902 | C1-TN5-7 | |||||
26 | STRT RIII NTC | 0.696 | No | N/A | Yes | 6 | 0 | C1-TN5-8 | |||||
27 | Smart2 RIII 1ng | 1.48 | No | Yes | 0.68 | 4.32 | S501/N701 | N/A | |||||
28 | Smart2 RIII 1ng | 1.47 | No | Yes | 0.68 | 4.32 | S502/N702 | N/A | |||||
29 | Smart2 RIII 100pg | 0.52 | No | Yes | 1.92 | 3.08 | S503/N703 | N/A | |||||
30 | Smart2 RIII 100pg | 0.49 | No | Yes | 2.04 | 2.96 | S504/N704 | N/A | |||||
31 | Smart2 RIII 10pg | 0.13 | No | Yes | 3.85 | 1.15 | S505/N705 | N/A | |||||
32 | Smart2 RIII 10pg | 0.12 | No | Yes | 4.17 | 0.83 | S506/N706 | N/A | |||||
33 | Smart2 RIII 10pg | - | No | Yes | 5.00 | 0.00 | S507/N707 | N/A | |||||
34 | Smart2 RIII NTC | - | No | Yes | 5.00 | 0.00 | S508/N708 | N/A | |||||
35 | Quartz RIII 1ng | 13.8 | No | Yes | 0.58 | 39.42 | S501/N709 | N/A | |||||
36 | Quartz RIII 1ng | 10.4 | No | Yes | 0.77 | 39.23 | S502/N710 | N/A | |||||
37 | Quartz RIII 100pg | 3.49 | No | Yes | 0.86 | 14.14 | S503/N711 | N/A | |||||
38 | Quartz RIII 100pg | 3.73 | No | Yes | 0.80 | 14.20 | S504/N712 | N/A | |||||
39 | Quartz RIII 10pg | 1.95 | No | Yes | 1.54 | 13.46 | S505/N701 | N/A | |||||
40 | Quartz RIII 10pg | 3.63 | No | Yes | 0.83 | 14.17 | S506/N702 | N/A | |||||
41 | Quartz RIII 10pg | 4.93 | No | Yes | 0.61 | 14.39 | S507/N703 | N/A | |||||
42 | Quartz RIII NTC | 1.83 | No | Yes | 1.64 | 13.36 | S508/N704 | N/A |
Nextera Tagmentation
Volume Tagment DNA Buffer 5ul Amplification Tagment Mix 2.5ul Diluted Sample 2.5ul Total 10ul
- Vortex and spin
- Incubate 55C 5 min
- Hold 10C
- Once sample reaches 10C add 2.5ul of NT buffer in block
- Vortex and spin
Nextera Amplification
Volume Tagmented Sample 12.5ul Nextera PCR Mix 7.5ul S5xx Primer 2.5ul S7xx Primer 2.5ul Total 20ul
- Mix and centrifuge
72C 3 min 95C 30s 95C 10s -> 55C 30s -> 72C 60s 12x 72C, 5min
- Run 2.5ul on gel:
File:03052014 PCR2 Gel1 Nextera.jpg
- Combined 5ul of each for Smart2
- Combined 5ul each of 1ng/100pg/NTC and 7.5ul each of 10pg for Quartz-seq
- Beads purified twice (0.8x volume) and resuspended in 10ul
Sten Tagmentation
Volume 5x Tagmentation Buffer 5ul Transposome stock (Epicenter) 6ul Sample 3ul dH2O 11ul Total 25ul
- Incubate @55C for 8 min
- Cool on ice
- Wash 100ul Dynabeads MyOne Streptavidin C1 Beads with 2xBWT and resuspended in 2ml 2xBWT
- Add 20ul beads to each well and incubate 5min
- Combine beads, immobilize and discard supernatant
- Wash beads with 100ul TNT
- Wash beads with 100ul Qiagen Qiaquick PB
- Wash beads twice with 100ul TNT
- Resuspend beads in 100ul restriction mix:
- 10ul NEBuffer 4
- 2ul PvuI-HF (0.4U/ul)
- 88ul dH2O
- Incubate at 37C for 1 hour
- Wash beads 3x with TNT
- Elute in 10ul 100mM NaOH, incubate 5 min then remove to new tube
- Neutralize by adding:
- 10ul 100mM HCl
- 5ul Neutralization buffer
- Check library size by gel (ran 5ul on UREA gel):
File:03052014 PCR2 Gel1 STRT.jpg
- Ran Sybr PCR using C1_P1_PCR_2/C1_TN5_U for 12 cycles (25ul total) and ran 5ul on gel: