Matthew Cai: Difference between revisions

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==Projects==
===in Situ Sequencing===
====Probe/Primer Design====
*[[Matt:LabNotes/exonProbeDesign|Padlock Probe Design]]
*[[Matt:LabNotes/2014-5-14|Designing Hexamer RT Primer Enriched in Targeted mRNA]]
====Probe Production and Testing====
*[[Matt:LabNotes/Probe Production and Capture|Probe Production and Capture]]
*[[Matt:LabNotes/2013-5-10|Agilent Probe Prep]]
*[[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_HL155:_Representation_Bias_of_Agi26k_Oligos_(Corrected)|Agi26k End Sequencing (HL155_130628) Analysis]]
*[[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====
*[[Matt:LabNotes/2014-5-7|ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA]]
=====Ampligase Efficiency Test=====
*[[Matt:LabNotes/2014-5-16|Detecting ppMALAT1 Hybridization]]


==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 ===
<calendar>
name=Matt
format=%name:LabNotes/%year-%month-%day
date=2015/01/01
view=oneyear
</calendar>
=== 2014 ===
=== 2014 ===
<calendar>
<calendar>
Line 34: Line 36:
view=oneyear
view=oneyear
</calendar>
</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===
*[[Matt:Experiments-FISSEQ|FISSEQ Experiments Page]]
====Probe/Primer Design====
*[[Matt:LabNotes/exonProbeDesign|Padlock Probe Design]]
*[[Matt:LabNotes/2014-9-19|Suppressor oligos for COL1A1]]
====Probe Production and Testing====
*[[Matt:LabNotes/Probe Production and Capture|CustomArray Probe Production and Capture]]
*[[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/2013-7-26#Analysis_of_HL152:_Representation_Bias_of_CA12k_Oligos_(Corrected)|CA12k End Sequencing (HL152_130524) Analysis]]
*[[Matt:LabNotes/2013-7-26#Analysis_of_HL155:_Representation_Bias_of_Agi26k_Oligos_(Corrected)|Agi26k End Sequencing (HL155_130628) Analysis]]
*[[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]]
===="Artificial" MALAT1 Rolony Experiments====
*[[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/2014-5-27#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/2014-5-16|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/2014-4-15|Ampligase Test Second Try]]
*[[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/2014-9-8|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====
*[[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==
Line 39: Line 238:
*[[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 47: 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<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==
CA12kNov2014_V4: 3,514 probes (150nt)<br>
CA12kNov2014_V6: 3,514 probes (150nt) (RevComp of V4)<br>
CA12kNov2014_V7: 2,486 probes (150nt)<br>
CA12kNov2014_V8: 2,486 probes (150nt) (RevComp of V7)<br>
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]

"Artificial" MALAT1 Rolony Experiments[edit]

Ampligase Efficiency Test[edit]

RT Primer Enrich mRNA -> cDNA[edit]

FISSEQ Experiments[edit]

Decoding[edit]


DARTFISH[edit]

Probe Design[edit]

Probe Prep[edit]

Probe Production[edit]

In vitro Capture[edit]

Dextran Sulfate + dcProbe[edit]

NGS of Rolonies[edit]

RNA-Seq of BA8[edit]

Fiducial Beads[edit]

VECTABOND[edit]

DARTFISH BA8[edit]

Validate with RNAscope BA8[edit]

Regression Analysis[edit]

RNA FISH + DARTFISH in Cultured Neurons[edit]

Mouse Embryo[edit]

Mouse Brain[edit]

SplintR in vitro Testing Additives ie Formamide[edit]

Image & Seq[edit]

Protocols[edit]

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