Hosuk:LabNotes/2014-8-19: Difference between revisions

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(Replaced content with "*LabNote ===Decoding with Agi26k0gap Padlock Probe=== ====Analysis==== *Image aligning *#Shift, Crop *#Shift *#BF ====Result==== *PGP1F_P20_S2_20...")
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===Decoding with Agi26k0gap Padlock Probe===
===Decoding with Agi26k0gap Padlock Probe===
*Human brain tissue from Yun
**HB3, section 9
**Total 15 tissue sections
*Mouse tissue : Total 15 tissue sections
*Stored in -80C




====Procedure====
====Analysis====
=====Fixation=====
*Image aligning
*Process
*#Shift, Crop
*#4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL)
*#Shift
*#*16% Paraformaldehyde Solution)
*#BF
*#*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
[[File:TissueGlassOnMatTek_2.png|550px]]
[[File:TissueGlassOnMatTek_1.png|400px]]


=====1st Rolony Process=====
*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.


 
====Result====
*Circligase II
*PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1
*#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
 
 
*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=====
*#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====
*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

Revision as of 20:49, 22 August 2014


Decoding with Agi26k0gap Padlock Probe

Analysis

  • Image aligning
    1. Shift, Crop
    2. Shift
    3. BF


Result

  • PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1