Matthew Cai
Notebook
2016
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2015
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2014
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2013
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Justin's Notebook
2015
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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
Fiducial Beads
VECTABOND
DARTFISH BA8
- 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
- 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
- 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
- DARTFISH + suppv2 of motor neurons + RNA FISH (KIT,CUX2,SNAP25)
- Design probes for 3 new genes (Never Ordered, switched to RNAscope instead)
Mouse Embryo
- 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
Mouse Brain
- FISSEQ tests of 3 pepsin incubation times: Attempt 1
- FISSEQ tests of 3 pepsin incubation times: Attempt 2
Protocols
- qPCR Protocol
- Bead Purification Protocol
- Qubit Protocol
- Polyacrylamide Gel Protocol
- CircLigase II Buffer
- BF "Skeleton" Image
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)
21 Decoding Probes