Matthew Cai: Difference between revisions
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==Notebook== | ==Notebook== | ||
=== 2017 === | |||
<calendar> | |||
name=Matt | |||
format=%name:LabNotes/%year-%month-%day | |||
date=2017/01/01 | |||
view=oneyear | |||
</calendar> | |||
=== 2016 === | |||
<calendar> | |||
name=Matt | |||
format=%name:LabNotes/%year-%month-%day | |||
date=2016/01/01 | |||
view=oneyear | |||
</calendar> | |||
=== 2015 === | === 2015 === | ||
<calendar> | <calendar> | ||
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format=%name:LabNotes/%year-%month-%day | format=%name:LabNotes/%year-%month-%day | ||
date=2013/01/01 | 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 | view=oneyear | ||
</calendar> | </calendar> | ||
==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]] | |||
*[[Matt:LabNotes/2015-10-19|CA12k_Nov2014 V4 Expansion PCR]] | |||
====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]] | |||
*[[Matt:LabNotes/2017-5-4|Agi15kFeb2017 V4 SplintR Capture with 20% Formamide]] | |||
*[[Matt:LabNotes/2017-6-19|Agi15kFeb2017 V4 SplintR Capture with 5% Formamide and 10% DMF]] | |||
====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]] | |||
*[[]] | |||
====Fiducial Beads==== | |||
*[[Matt:LabNotes/2015-8-3|Choosing buffer]] | |||
*[[Matt:LabNotes/2015-8-4|DARTFISH BA8 with 1:500 Fiducial beads]] | |||
*[[Matt:LabNotes/2015-8-13|BA8 with 1:2000 Fiducial beads]] | |||
====VECTABOND==== | |||
*[[Matt:LabNotes/2015-9-25|Treat 50 coverslips and 20 slides]] | |||
====DARTFISH BA8==== | |||
*[[Matt:LabNotes/2015-8-17|DARTFISH suppv2 BA8 with Fiducial beads failed because frozen/thawed PFA]] | |||
*[[Matt:LabNotes/2015-8-18|DARTFISH suppv2 BA8 with Fiducial beads 0.3um z-stepsize 6 positions for 3D Decoding]] | |||
*[[Matt:LabNotes/2015-9-2|Decode BA8 V4 Sample made by Hosuk with Fiducial beads 0.3um z-stepsize 4 positions for 3D Decoding]] | |||
*[[Matt:LabNotes/2015-9-29|DARTFISH and FISSEQ on glass slides for Harvard to decode/sequence]] | |||
*[[Matt:LabNotes/2015-10-7#FISSEQ_on_BA8_for_Harvard|FISSEQ on glass slide for Harvard to sequence]] | |||
*[[Matt:LabNotes/2015-10-12|DARTFISH suppv2 BA8]] | |||
*[[Matt:LabNotes/2015-10-20|DARTFISH w/&w/o suppv2 BA8 with Fiducial]] | |||
*[[Matt:LabNotes/2015-10-29|DARTFISH suppv2 BA8 90sec 0.01% pepsin]] | |||
====Validate with RNAscope BA8==== | |||
*[[Matt:LabNotes/2015-11-6|RNAscope of BA8: RELN, SLC17A7, PDE1A, OLFM1]] | |||
*[[Matt:LabNotes/2015-11-11|20X DARTFISH Imaging of 'DARTFISH suppv2 BA8 90sec 0.01% pepsin' Tile]] | |||
*[[Matt:LabNotes/2015-11-15|20X DARTFISH Analysis of 'DARTFISH suppv2 BA8 90sec 0.01% pepsin' Tile]] | |||
*[[Matt:LabNotes/2015-12-11|20X DARTFISH DE & Subpopulation Analysis]] | |||
====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]] | |||
*[[Matt:LabNotes/2015-7-20|DARTFISH + suppv2 of motor neurons + RNA FISH (KIT,CUX2,SNAP25)]] | |||
*[[Matt:LabNotes/2015-7-21|Design probes for 3 new genes (Never Ordered, switched to RNAscope instead)]] | |||
===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]] | |||
*[[Matt:LabNotes/2015-10-7#FISSEQ_on_Mouse_Embryo_Test|FISSEQ on mouse embryo section Try 1]] | |||
*[[Matt:LabNotes/2015-12-14|FISSEQ on mouse embryo section Try 2]] | |||
*[[Matt:LabNotes/2016-3-8|FISSEQ attempt 4 in whole mouse embryo]] | |||
*[[Matt:LabNotes/2016-5-17|FISSEQ attempt 5 in whole mouse embryo]] | |||
*[[Matt:LabNotes/2016-6-15|FISSEQ attempt 6 in whole mouse embryo]] | |||
*[[Matt:LabNotes/2016-7-19|FISSEQ attempt 7 with PACT/CLARITY in whole mouse embryo]] | |||
*[[Matt:LabNotes/2016-9-28|FISSEQ attempt 8 with Focus Clear]] | |||
===Mouse Brain=== | |||
*[[Matt:LabNotes/2016-8-17|FISSEQ tests of 3 pepsin incubation times: Attempt 1]] | |||
*[[Matt:LabNotes/2016-8-18|FISSEQ tests of 3 pepsin incubation times: Attempt 2]] | |||
===SplintR in vitro Testing Additives ie Formamide=== | |||
*[[Matt:LabNotes/2017-4-22|1st Try]] | |||
*[[Matt:LabNotes/2017-5-8|2nd Try: ET SSB + 10% Formamide]] | |||
*[[Matt:LabNotes/2017-5-15|3rd Try]] | |||
*[[Matt:LabNotes/2017-5-19|4th Try]] | |||
*[[Matt:LabNotes/2017-5-22|5th Try]] | |||
*[[Matt:LabNotes/2017-6-7|6th Try: DMF, DMSO, Betaine]] | |||
*[[Matt:LabNotes/2017-5-4|Agi15kFeb2017 V4 SplintR Capture with 20% Formamide]] | |||
*[[Matt:LabNotes/2017-6-19|Agi15kFeb2017 V4 SplintR Capture with 5% Formamide and 10% DMF]] | |||
===Image & Seq=== | |||
*[[Matt:LabNotes/2017-6-1|1st Try]] | |||
*[[Matt:LabNotes/2017-6-8|2nd Try: Vary number of cycles]] | |||
*[[Matt:LabNotes/2017-6-13|3rd Try: USER]] | |||
*[[Matt:LabNotes/2017-6-14|4th Try: USER]] | |||
*[[Matt:LabNotes/2017-6-27|5th Try: USER, 45C Annealing, with Magnet]] | |||
==Protocols== | ==Protocols== | ||
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*[[Matt:LabNotes/Bead Purification Protocol|Bead Purification Protocol]] | *[[Matt:LabNotes/Bead Purification Protocol|Bead Purification Protocol]] | ||
*[[Matt:LabNotes/Qubit Protocol|Qubit Protocol]] | *[[Matt:LabNotes/Qubit Protocol|Qubit Protocol]] | ||
*[[Matt:LabNotes/Polyacrylamide Gel Protocol|Polyacrylamide Gel Protocol]] | |||
*[[Hosuk:LabNotes/2014-3-24|CircLigase II Buffer]] | |||
*[[Matt:LabNotes/2014-2-19|BF "Skeleton" Image]] | *[[Matt:LabNotes/2014-2-19|BF "Skeleton" Image]] | ||
Line 66: | Line 247: | ||
130628_HL155 (Lane 3, Indx 10 & 12): Agi26k_0gap and Agi26k_20gap oligos<br> | 130628_HL155 (Lane 3, Indx 10 & 12): Agi26k_0gap and Agi26k_20gap oligos<br> | ||
130729_MiSeq: Agi26k_0gap and Agi26k_20gap capture of gDNA and cDNA<br> | 130729_MiSeq: Agi26k_0gap and Agi26k_20gap capture of gDNA and cDNA<br> | ||
131220_HL162 (Lane 1, unassigned): PhiX Control | 131220_HL162 (Lane 1, unassigned): PhiX Control<br> | ||
150602_MiSeq: CA12kNov14suppv2_gDNA and CA12kNov14suppv2_cDNAdT<br> | |||
150623_MiSeq: CA12kNov14suppv2_cDNARan and CA12kNov14suppv2_NegCtrl<br> | |||
150616_MiSeq: [[Matt:LabNotes/2015-5-26 | SMART-Seq of whole BA8 tissue section]]<br> | |||
==Probe Sets== | ==Probe Sets== | ||
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CA12k: 12,355 probes (170nt)<br> | CA12k: 12,355 probes (170nt)<br> | ||
Agi26k_0gap: 12,964 probes (193nt) <br> | Agi26k_0gap: 12,964 probes (193nt) <br> | ||
Agi26k_20gap: 13,179 probes (193nt) | Agi26k_20gap: 13,179 probes (193nt) <br> | ||
[[Matt:LabNotes/dcProbes7x3 |21 Decoding Probes]] |
Latest revision as of 02:48, 12 July 2017
Notebook[edit]
2017[edit]
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2017/01/01 view=oneyear </calendar>
2016[edit]
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2016/01/01 view=oneyear </calendar>
2015[edit]
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2015/01/01 view=oneyear </calendar>
2014[edit]
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2014/01/01 view=oneyear </calendar>
2013[edit]
<calendar> name=Matt format=%name:LabNotes/%year-%month-%day date=2013/01/01 view=oneyear </calendar>
Justin's Notebook[edit]
2015[edit]
<calendar> name=Matt format=%name:JustinLabNotes/%year-%month-%day date=2015/01/01 view=oneyear </calendar>
Projects[edit]
2 Step ppCapture + RCA modified FISSEQ[edit]
Probe/Primer Design[edit]
Probe Production and Testing[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
Decoding[edit]
DARTFISH[edit]
Probe Design[edit]
- 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[edit]
Probe Production[edit]
- 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[edit]
- 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
- Agi15kFeb2017 V4 SplintR Capture with 20% Formamide
- Agi15kFeb2017 V4 SplintR Capture with 5% Formamide and 10% DMF
Dextran Sulfate + dcProbe[edit]
NGS of Rolonies[edit]
RNA-Seq of BA8[edit]
Fiducial Beads[edit]
VECTABOND[edit]
DARTFISH BA8[edit]
- DARTFISH suppv2 BA8 with Fiducial beads failed because frozen/thawed PFA
- DARTFISH suppv2 BA8 with Fiducial beads 0.3um z-stepsize 6 positions for 3D Decoding
- Decode BA8 V4 Sample made by Hosuk with Fiducial beads 0.3um z-stepsize 4 positions for 3D Decoding
- DARTFISH and FISSEQ on glass slides for Harvard to decode/sequence
- FISSEQ on glass slide for Harvard to sequence
- DARTFISH suppv2 BA8
- DARTFISH w/&w/o suppv2 BA8 with Fiducial
- DARTFISH suppv2 BA8 90sec 0.01% pepsin
Validate with RNAscope BA8[edit]
- RNAscope of BA8: RELN, SLC17A7, PDE1A, OLFM1
- 20X DARTFISH Imaging of 'DARTFISH suppv2 BA8 90sec 0.01% pepsin' Tile
- 20X DARTFISH Analysis of 'DARTFISH suppv2 BA8 90sec 0.01% pepsin' Tile
- 20X DARTFISH DE & Subpopulation Analysis
Regression Analysis[edit]
- 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[edit]
- 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
- DARTFISH + suppv2 of motor neurons + RNA FISH (KIT,CUX2,SNAP25)
- Design probes for 3 new genes (Never Ordered, switched to RNAscope instead)
Mouse Embryo[edit]
- FISSEQ attempt 1 in whole mouse embryo
- FISSEQ attempt 2 in whole mouse embryo
- FISSEQ attempt 3 in whole mouse embryo
- FISSEQ on mouse embryo section Try 1
- FISSEQ on mouse embryo section Try 2
- FISSEQ attempt 4 in whole mouse embryo
- FISSEQ attempt 5 in whole mouse embryo
- FISSEQ attempt 6 in whole mouse embryo
- FISSEQ attempt 7 with PACT/CLARITY in whole mouse embryo
- FISSEQ attempt 8 with Focus Clear
Mouse Brain[edit]
- FISSEQ tests of 3 pepsin incubation times: Attempt 1
- FISSEQ tests of 3 pepsin incubation times: Attempt 2
SplintR in vitro Testing Additives ie Formamide[edit]
- 1st Try
- 2nd Try: ET SSB + 10% Formamide
- 3rd Try
- 4th Try
- 5th Try
- 6th Try: DMF, DMSO, Betaine
- Agi15kFeb2017 V4 SplintR Capture with 20% Formamide
- Agi15kFeb2017 V4 SplintR Capture with 5% Formamide and 10% DMF
Image & Seq[edit]
- 1st Try
- 2nd Try: Vary number of cycles
- 3rd Try: USER
- 4th Try: USER
- 5th Try: USER, 45C Annealing, with Magnet
Protocols[edit]
- qPCR Protocol
- Bead Purification Protocol
- Qubit Protocol
- Polyacrylamide Gel Protocol
- CircLigase II Buffer
- BF "Skeleton" Image
Sequencing Runs[edit]
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[edit]
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)
21 Decoding Probes