Matthew Cai: Difference between revisions
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==Projects== | ==Projects== | ||
=== | ===2 Step ppCapture + RCA modified FISSEQ=== | ||
*[[Matt:Experiments-FISSEQ|FISSEQ Experiments Page]] | *[[Matt:Experiments-FISSEQ|FISSEQ Experiments Page]] | ||
====Probe/Primer Design==== | ====Probe/Primer Design==== | ||
*[[Matt:LabNotes/exonProbeDesign|Padlock Probe Design]] | *[[Matt:LabNotes/exonProbeDesign|Padlock Probe Design]] | ||
*[[Matt:LabNotes/2014- | *[[Matt:LabNotes/2014-9-19|Suppressor oligos for COL1A1]] | ||
====Probe Production and Testing==== | ====Probe Production and Testing==== | ||
*[[Matt:LabNotes/Probe Production and Capture|CustomArray Probe Production and Capture]] | *[[Matt:LabNotes/Probe Production and Capture|CustomArray Probe Production and Capture]] | ||
*[[Matt:LabNotes/2013-5-10|Agilent Probe Prep]] | *[[Matt:LabNotes/2013-5-10|Agilent Probe Prep]] | ||
*[[Matt:LabNotes/2014-5-22|Agi26k0gap Probe Production]] | |||
*[[Matt:LabNotes/2014-6-11|Agi26k0gap Probe Production]] | |||
*[[Matt:LabNotes/2014-7-1|Agi26k0gap Probe Production]] | |||
*[[Matt:LabNotes/2014-8-15|Agi26k0gap Probe Production]] | |||
*[[Matt:LabNotes/2014-9-18|Agi26k0gap Probe Production]] | |||
*[[Matt:LabNotes/2013-8-20|CA12k Capture (MiSeq_130325) Analysis]] | *[[Matt:LabNotes/2013-8-20|CA12k Capture (MiSeq_130325) Analysis]] | ||
*[[Matt:LabNotes/2013-7-26#Analysis_of_HL152:_Representation_Bias_of_CA12k_Oligos_(Corrected)|CA12k End Sequencing (HL152_130524) Analysis]] | *[[Matt:LabNotes/2013-7-26#Analysis_of_HL152:_Representation_Bias_of_CA12k_Oligos_(Corrected)|CA12k End Sequencing (HL152_130524) Analysis]] | ||
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*[[Matt:LabNotes/2013-8-9#Quantifying_Errors_in_CA12k_and_Agi26k_Oligo_Pools_.28Ignoring_low_base_quality_substitution_errors.29|Quantifying Errors in CA12k and Agi26k Oligo Pools]] | *[[Matt:LabNotes/2013-8-9#Quantifying_Errors_in_CA12k_and_Agi26k_Oligo_Pools_.28Ignoring_low_base_quality_substitution_errors.29|Quantifying Errors in CA12k and Agi26k Oligo Pools]] | ||
====Rolony Experiments==== | ===="Artificial" MALAT1 Rolony Experiments==== | ||
*[[Matt:LabNotes/2014-4-30|Making artificial MALAT1 rolonies (100nM template -> 10pM ppMALAT1)]] | *[[Matt:LabNotes/2014-4-30|Making artificial MALAT1 rolonies (100nM template -> 10pM ppMALAT1)]] | ||
*[[Matt:LabNotes/2014-5-13|Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1)]] | *[[Matt:LabNotes/2014-5-13|Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1)]] | ||
*[[Matt:LabNotes/2014-5-27#Artifical_Rolonies|Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1) trial with better cells]] | *[[Matt:LabNotes/2014-5-27#Artifical_Rolonies|Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1) trial with better cells]] | ||
*[[Matt:LabNotes/2014-5-7|ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA]] | *[[Matt:LabNotes/2014-5-7|ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA]] | ||
*[[Matt:LabNotes/2014-5-16|Detecting captured padlock probes]] | |||
*[[Matt:LabNotes/2014-9-6|Tertiary Rolony synthesis]] | |||
====Ampligase Efficiency Test==== | |||
*[[Matt:LabNotes/2014-4-9|Ampligase Test First Try]] | *[[Matt:LabNotes/2014-4-9|Ampligase Test First Try]] | ||
*[[Matt:LabNotes/2014-4-15|Ampligase Test Second Try]] | *[[Matt:LabNotes/2014-4-15|Ampligase Test Second Try]] | ||
*[[Matt:LabNotes/2014-5-16|Detecting ppMALAT1 Hybridization]] | *[[Matt:LabNotes/2014-5-16|Detecting ppMALAT1 Hybridization]] | ||
*[[Matt:LabNotes/2014-5-27#Exo_I.2FIII_Test|Testing Exo I/III Digestion of Hybridized Padlock Probes]] | *[[Matt:LabNotes/2014-5-27#Exo_I.2FIII_Test|Testing Exo I/III Digestion of Hybridized Padlock Probes]] | ||
*[[Matt:LabNotes/2014-9-8|45C vs 60C Ampligase Incubation]] | |||
====RT Primer Enrich mRNA -> cDNA==== | |||
*[[Matt:LabNotes/2014-5-14|Designing Hexamer RT Primer Enriched in Targeted mRNA]] | |||
*[[Matt:LabNotes/2014-6-9|Top48 RT Primer in vitro Validation shows UHRR has DNA contamination]] | |||
*[[Matt:LabNotes/2014-6-18|Repeat Top48 Hexamer RT Primer in vitro Validation]] | |||
*[[Matt:LabNotes/2014-7-14|Analyzing in vitro RNA-Seq with RT Primers]] | |||
*[[Matt:LabNotes/2014-7-30|Analyzing in vitro RNA-Seq with RT Primers continued]] | |||
*[[Matt:LabNotes/2014-8-1|Analyzing in vitro RNA-Seq with RT Primers continued]] | |||
*[[Matt:LabNotes/2014-9-5|qMDA confirms UHRR DNA contamination]] | |||
*[[Matt:LabNotes/2014-9-25|Top48 RT Primer in vitro Validation with purified UHRR]] | |||
*[[Matt:LabNotes/2014-10-31|RT Primer RNA-Seq Analysis]] | |||
====FISSEQ Experiments==== | |||
*[[Matt:LabNotes/2014-10-6|Agi26k0gap + suppressor oligos & Agi26k20gap on PGP1f]] | |||
====Decoding==== | ====Decoding==== | ||
*[[Matt:LabNotes/2014-4-16|Decoding Partial Barcode (only 10 dye probes)]] | *[[Matt:LabNotes/2014-4-16|Decoding Partial Barcode (only 10 dye probes)]] | ||
*[[Matt:LabNotes/2014-8-20|First Full Decoding: PGP1F_Agi26k0gap with PISA Mask]] | |||
===DARTFISH=== | |||
*[[Matt:LabNotes/2014-9-2|Barcoding Scheme]] | |||
====Probe Design==== | |||
*[[Matt:LabNotes/2014-9-17|Gene selection]] | |||
*[[Matt:LabNotes/2014-10-9|Design New Padlock Probe Set]] | |||
*[[Matt:LabNotes/2014-10-30|ppDesigner on 450 genes]] | |||
*[[Matt:LabNotes/2014-11-1|ppDesigner on new genelist]] | |||
*[[Matt:LabNotes/2014-11-10|ppDesigner on new genelist + contigs to meet 12,000 oligo requirement]] | |||
*[[Matt:LabNotes/2014-11-19|Final Padlock Probe Design: CA12k_Nov2014]] | |||
====Probe Prep==== | |||
*[[Matt:LabNotes/2014-12-18|CA12k_Nov2014 Expansion PCR Test]] | |||
====Probe Production==== | |||
*[[Matt:LabNotes/2015-1-5|CA12k_Nov2014 V4 and V7 Probe Production]] | |||
*[[Matt:LabNotes/2015-1-13|CA12k_Nov2014 V7 Probe Production]] | |||
*[[Matt:LabNotes/2015-1-15|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-1-26|CA12k_Nov2014 V6 and V8 Probe Production]] | |||
*[[Matt:LabNotes/2015-2-2|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-2-5|CA12k_Nov2014 V7 Probe Production]] | |||
*[[Matt:LabNotes/2015-2-28|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-3-25|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-5-5|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-5-11|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-6-3|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-6-29|CA12k_Nov2014 V4 Probe Production]] | |||
*[[Matt:LabNotes/2015-5-11|CA12k_Nov2014 V4 Probe Production]] | |||
====In vitro Capture==== | |||
*[[Matt:LabNotes/2015-1-12|UHRR cDNA synthesis]] | |||
*[[Matt:LabNotes/2015-1-21|V4 and V7 Capture]] | |||
*[[Matt:LabNotes/2015-3-19|V4 Capture Sequencing Analysis + Design 39 suppressor oligos]] | |||
*[[Matt:LabNotes/2015-4-8|V7 Capture Sequencing Analysis]] | |||
*[[Matt:LabNotes/2015-4-28|V4 + supp oligo Capture]] | |||
*[[Matt:LabNotes/2015-5-8|V4 + supp oligo Capture Sequencing Analysis]] | |||
*[[Matt:LabNotes/2015-5-15|BA8 cDNA synthesis]] | |||
*[[Matt:LabNotes/2015-5-18|V4 + suppv2 oligo Capture]] | |||
*[[Matt:LabNotes/2015-7-12|V4 + suppv2 oligo Capture Sequencing Analysis]] | |||
====Dextran Sulfate + dcProbe==== | |||
*[[Matt:LabNotes/2015-3-12|Dextran sulfate to improve fluorescence intensity distribution -> bimodal]] | |||
====NGS of Rolonies==== | |||
*[[Matt:LabNotes/2015-3-14|Primer design]] | |||
*[[Matt:LabNotes/2015-3-31|Amplify rolonies via slide PCR Experiment]] | |||
*[[Matt:LabNotes/2015-4-13|Sequencing alignment and analysis]] | |||
====RNA-Seq of BA8==== | |||
*[[Matt:LabNotes/2015-5-19|RNA-Seq not sensitive enough for small amounts of isolated RNA]] | |||
*[[Matt:LabNotes/2015-5-26|SMARTer_Seq]] | |||
====Regression Analysis==== | |||
*[[Matt:LabNotes/2015-4-8#Regression_Analysis|Px-px decoding of DARTFISH PGP1f & BA8]] | |||
*[[Matt:LabNotes/2015-4-14|Try normalize DARTFISH with in vitro cDNA capture]] | |||
*[[Matt:LabNotes/2015-4-15|Spearman's rank correlation]] | |||
*[[Matt:LabNotes/2015-5-14|V4 + supp oligos normalized vs HBRR/UHRR]] | |||
*[[]] | |||
===RNA FISH + DARTFISH in Cultured Neurons=== | |||
*[[Matt:LabNotes/2015-6-5|Probe Resuspension (ADARB2,CUX2,SATB2,SLC6A1) and Dye Coupling(SLC6A1,SATB2)]] | |||
*[[Matt:LabNotes/2015-6-8|RNA FISH & DARTFISH & FISSEQ in iPS derived motor neurons from Yeo lab]] | |||
*[[Matt:LabNotes/2015-6-17|Decoded DARTFISH of iPS derived motor neurons]] | |||
*[[Matt:LabNotes/2015-6-19|DARTFISH + suppv2 of iPS derived motor neurons]] | |||
*[[Matt:LabNotes/2015-6-15|Probe Resuspension (KIT,SNAP25) and Dye Coupling(KIT,SNAP25)]] | |||
*[[Matt:LabNotes/2015-6-18|RNA FISH (KIT,CUX2) in iPS derived motor neurons from Yeo lab]] | |||
*[[Matt:LabNotes/2015-6-30|RNA FISH (KIT,CUX2) in iPS derived motor neurons from Yeo lab with cooled CCD]] | |||
*[[Matt:LabNotes/2015-7-1|DARTFISH + suppv2 of iNGN from Harvard]] | |||
*[[Matt:LabNotes/2015-7-8|RNA FISH (CUX2 even and odd) in iNGN from Harvard]] | |||
*[[Matt:LabNotes/2015-7-14|Improve dye coupling by repeating column purification]] | |||
===Mouse Embryo=== | |||
*[[Matt:LabNotes/2015-6-23|FISSEQ attempt 1 in whole mouse embryo]] | |||
*[[Matt:LabNotes/2015-7-6|FISSEQ attempt 2 in whole mouse embryo]] | |||
*[[Matt:LabNotes/2015-7-7|FISSEQ attempt 3 in whole mouse embryo]] | |||
==Protocols== | ==Protocols== |
Revision as of 23:10, 20 October 2015
Notebook
2015
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2015/01/01 view=oneyear </calendar>
2014
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2014/01/01 view=oneyear </calendar>
2013
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2013/01/01 view=oneyear </calendar>
Justin's Notebook
2015
<calendar> name=Matt format=%name:JustinLabNotes/%year-%month-%day date=2015/01/01 view=oneyear </calendar>
Projects
2 Step ppCapture + RCA modified FISSEQ
Probe/Primer Design
Probe Production and Testing
- CustomArray Probe Production and Capture
- Agilent Probe Prep
- Agi26k0gap Probe Production
- Agi26k0gap Probe Production
- Agi26k0gap Probe Production
- Agi26k0gap Probe Production
- Agi26k0gap Probe Production
- CA12k Capture (MiSeq_130325) Analysis
- CA12k End Sequencing (HL152_130524) Analysis
- Agi26k End Sequencing (HL155_130628) Analysis
- Quantifying Errors in CA12k and Agi26k Oligo Pools
"Artificial" MALAT1 Rolony Experiments
- Making artificial MALAT1 rolonies (100nM template -> 10pM ppMALAT1)
- Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1)
- Making artificial MALAT1 rolonies (100nM template -> 100pM ppMALAT1) trial with better cells
- ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA
- Detecting captured padlock probes
- Tertiary Rolony synthesis
Ampligase Efficiency Test
- Ampligase Test First Try
- Ampligase Test Second Try
- Detecting ppMALAT1 Hybridization
- Testing Exo I/III Digestion of Hybridized Padlock Probes
- 45C vs 60C Ampligase Incubation
RT Primer Enrich mRNA -> cDNA
- Designing Hexamer RT Primer Enriched in Targeted mRNA
- Top48 RT Primer in vitro Validation shows UHRR has DNA contamination
- Repeat Top48 Hexamer RT Primer in vitro Validation
- Analyzing in vitro RNA-Seq with RT Primers
- Analyzing in vitro RNA-Seq with RT Primers continued
- Analyzing in vitro RNA-Seq with RT Primers continued
- qMDA confirms UHRR DNA contamination
- Top48 RT Primer in vitro Validation with purified UHRR
- RT Primer RNA-Seq Analysis
FISSEQ Experiments
Decoding
DARTFISH
Probe Design
- Gene selection
- Design New Padlock Probe Set
- ppDesigner on 450 genes
- ppDesigner on new genelist
- ppDesigner on new genelist + contigs to meet 12,000 oligo requirement
- Final Padlock Probe Design: CA12k_Nov2014
Probe Prep
Probe Production
- CA12k_Nov2014 V4 and V7 Probe Production
- CA12k_Nov2014 V7 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V6 and V8 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V7 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
- CA12k_Nov2014 V4 Probe Production
In vitro Capture
- UHRR cDNA synthesis
- V4 and V7 Capture
- V4 Capture Sequencing Analysis + Design 39 suppressor oligos
- V7 Capture Sequencing Analysis
- V4 + supp oligo Capture
- V4 + supp oligo Capture Sequencing Analysis
- BA8 cDNA synthesis
- V4 + suppv2 oligo Capture
- V4 + suppv2 oligo Capture Sequencing Analysis
Dextran Sulfate + dcProbe
NGS of Rolonies
RNA-Seq of BA8
Regression Analysis
- Px-px decoding of DARTFISH PGP1f & BA8
- Try normalize DARTFISH with in vitro cDNA capture
- Spearman's rank correlation
- V4 + supp oligos normalized vs HBRR/UHRR
- [[]]
RNA FISH + DARTFISH in Cultured Neurons
- Probe Resuspension (ADARB2,CUX2,SATB2,SLC6A1) and Dye Coupling(SLC6A1,SATB2)
- RNA FISH & DARTFISH & FISSEQ in iPS derived motor neurons from Yeo lab
- Decoded DARTFISH of iPS derived motor neurons
- DARTFISH + suppv2 of iPS derived motor neurons
- Probe Resuspension (KIT,SNAP25) and Dye Coupling(KIT,SNAP25)
- RNA FISH (KIT,CUX2) in iPS derived motor neurons from Yeo lab
- RNA FISH (KIT,CUX2) in iPS derived motor neurons from Yeo lab with cooled CCD
- DARTFISH + suppv2 of iNGN from Harvard
- RNA FISH (CUX2 even and odd) in iNGN from Harvard
- Improve dye coupling by repeating column purification
Mouse Embryo
- FISSEQ attempt 1 in whole mouse embryo
- FISSEQ attempt 2 in whole mouse embryo
- FISSEQ attempt 3 in whole mouse embryo
Protocols
Sequencing Runs
130325_MiSeq: CA12k capture of gDNA, cDNAwRNase, and cDNA-RNase
130524_HL152 (Lane 2, unassigned): CA12k oligos
130628_HL155 (Lane 3, Indx 10 & 12): Agi26k_0gap and Agi26k_20gap oligos
130729_MiSeq: Agi26k_0gap and Agi26k_20gap capture of gDNA and cDNA
131220_HL162 (Lane 1, unassigned): PhiX Control
150602_MiSeq: CA12kNov14suppv2_gDNA and CA12kNov14suppv2_cDNAdT
150623_MiSeq: CA12kNov14suppv2_cDNARan and CA12kNov14suppv2_NegCtrl
150616_MiSeq: SMART-Seq of whole BA8 tissue section
Probe Sets
CA12kNov2014_V4: 3,514 probes (150nt)
CA12kNov2014_V6: 3,514 probes (150nt) (RevComp of V4)
CA12kNov2014_V7: 2,486 probes (150nt)
CA12kNov2014_V8: 2,486 probes (150nt) (RevComp of V7)
CA12k: 12,355 probes (170nt)
Agi26k_0gap: 12,964 probes (193nt)
Agi26k_20gap: 13,179 probes (193nt)