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==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]]
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