Hosuk:LabNotes/2014-8-18
Jump to navigation
Jump to search
Decoding with Agi26k0gap Padlock Probe[edit]
- Human brain tissue from Yun
- HB3, section 9
- Total 15 tissue sections
- Mouse tissue : Total 15 tissue sections
- Stored in -80C
Procedure[edit]
Fixation[edit]
- Process
- 4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL)
- 16% Paraformaldehyde Solution)
- Manufacture : Electron Microscopy Diatome --> 16% PARAFORMALDEHYDE AQ SOLUTN 50-980-486, No.:15700
- Supply : (Fisher Scientific), 50980486
- Paraformaldehyde - methanol free solution. A more efficient and rapid penetrant fixative used in combination with Glutaraldehyde and Acrolein fixatives. Easy to break, prescored, 10 ml ampoules sealed under inert gas 10 ml
- Take Tissue glass slide from -80C, and put them to room temp. for 2min.
- Add 4% formamide on a tissue, incubate for 30min at room temp.
- Wash with PBS 3 times (add PBS in 3 wells of 6 well plate and series washing with glasses)
- Attach glass with glue
- Cyanoacrylate adhesives : High-viscosity, Sigma-Aldrich, Z105902-1EA
- Wipe PBS from the glass (around tissue)
- Apply glue along the edge of the glass, put on the bottom of MatTek dish, wait for 5min
- 4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL)
File:TissueGlassOnMatTek 2.png File:TissueGlassOnMatTek 1.png
1st Rolony Process[edit]
- RT
- Wash with H2O for 5min. twice
- Add 0.25% TX-100 + DEPC-H2O, incubate for 15min. at room temp.
- Wash with DEPC-H2O twice
- Add 200 ul 0.1% pepsin in 0.1 N HCl (4uL of 5% Pepsin + 10uL of 2N HCl + 186uL DEPC-H2O), incubate for 10 min at 37C
- Wash with DEPC-PBS three times to inactivate pepsin
- Add RT mix (Hexamer, -a-dUTP)
- DEPC-H2O 161uL
- M-MuLV Buffer 20uL
- 25mM dNTP 2uL
- 100uM random hexamer RT Primer 5uL
- Rnase Inhibitor 2uL
- M-MuLV reverse transcriptase 10 uL
- Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.
- Circligase II
- Wash with PBS once, skip BS(PEG)9
- Add RNase H mix, incubate for 1hr. at 37C
- H2O 168uL
- Riboshredder 2uL
- RNase H buffer 20uL
- RNase H 10uL
- Wash with nucleaus-free H2O twice
- Add CircLigase II mix, incubate at 60C for 2hr.
- H2O 128uL
- Buffer 20uL
- 50mM MnCl2 10uL
- 5M Betaine 40uL
- CircLigaseII 2uL
- RCA
- Add 0.5uM RCA Primer in 200uL (2x SSC + 30% Formamide), incubate at 60C for 1hr.
- 1uL of 100uM RCA Primer + 199uL (2X SSC + 30% Formamide)
- Wash with 2x SSC, 1x SSC twice each
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29(100 U/µl) 2uL
- Add RCA mix, incubate at 30C for overnight
- Add 0.5uM RCA Primer in 200uL (2x SSC + 30% Formamide), incubate at 60C for 1hr.
- Post fix
- Wash with PBS once
- add 200uL BS(PEG)9 mix (4uL BS(PEG)9 in 196uL 1x PBS), incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- Wash with PBS once
- Imaging
- Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
- Preheat detection Probe at 80C for 5min
- Add to sample and incubate at RT for 10min
- Wash two times for 1 minute each with 1 mL 1X PBS
Padlock Probe capture, 2nd Rolony Process[edit]
- Strip Cy3-1st Rolony detection Probes with preheated 80% formamide at 70C, incubate for 15min
- Wash with PBS 3 times
- Prepare Agi26k0gap : 50nM in 120uL (Matt's product) --> 30uL in 100uL 2x SSC, which is 15nM
- 30uL Agi26k0gap (15nM)
- 10uL Ampligase (5 U/uL)
- 10uL Buffer
- 50uL H2O
- Add 100uL per MatTek dish, incubate dishes at 37C for 30min, and 45C for overnight
- Wash with PBS twice,
- Preheat 0.5uM FISSEQ_ppRCA primer in 2X SSC at 70C, and add and incubate it at 45C for 30min
- Wash with PBS twice
- Add RCA mix, incubate at 30C for overnight (start : 08/12 3pm)
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (100 U/µl) : 2uL
- Wash with PBS once, add BS(PEG)9, incubate at room temp. for 1hr.
- Wash with PBS twice, add 1M Tris pH8.0 , incubate at room temp. for 1hr.
- Wash with PBS twice, store at PBS
Decoding Procedure[edit]
- dcProbe set : 0.5uM each dcProbe in 100uL(2x SSC + 30% Formamide)
- 5uL of 10uM dcProbe#_488 + 5uL of 10uM dcProbe#_Cy3 + 5uL of 10uM dcProbe#_Cy5 + 85uL of (2xSSC + 80% Formamide)
- Procedure
- Image 1st Rolony : FISSEQ-Adapt, Cy3, and find proper positions for decoding, Mark positions
- Aspirate PBS and Strip with preheated 80% Formamide(15min incubation), wash with PBS 3 times
- Add dcProbe0 mix, incubate for 10min., wash with 2x SSC twice
- Image, ch00 --> 488, ch01 --> Cy3, ch02 --> Cy5, ch03 --> bright field
- Aspirate PBS and Strip with preheated 80% Formamide(15min incubation), wash with PBS 3 times
- Add dcProbe1 mix, incubate for 10min., wash with 2x SSC twice
- Image, ... repeat until dcProbe7
Samples[edit]
Status[edit]
- Finish 2nd Rolony : Human Tissue section --> HBTissue_2014-08-12_S1, HBTissue_2014-08-12_S2
- Finish 2nd Rolony : PGP1F –-> PGP1F_P16_S1_2014-08-09, PGP1F_P20_S2_2014-08-09
- Finish Decoding : PGP1F_P20_S2_2014-08-09, HBTissue_2014-08-12_S1
Result[edit]
- HBTissue_2014-08-12_S1 : Most of signal seemed non-specific binding, not from real rolonies.
- PGP1F_P20_S2_2014-08-09 --> performed 7 cycle run (--> Date : 2014-08-13)
- Analysis result will be on 08-19