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===Decoding with Agi26k0gap Padlock Probe===
===Decoding with Agi26k0gap Padlock Probe===
*Human brain tissue from Yun
*Image resolution : 20x obj, 4096 x 4086, z step = 1.04um
**HB3, section 9
**Since how many features would be detected or how much dense rolonies in this sample, so image in wide field of view
**Total 15 tissue sections
**eventually 63x obj.
*Mouse tissue : Total 15 tissue sections
*Data is in '2014-08-13'
*Stored in -80C




====Procedure====
====Analysis====
=====Fixation=====
*1. Arrange data
*Process
*#File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif
*#4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL)
*#*ex) PGP1F : PGP1F_Agi26k0gap_Decode_P20_S2_2014-08-09_20x_4k_Step1_Pos2_z01_ch01.tif
*#*16% Paraformaldehyde Solution)
*#*ex) Tissue section : HBTissue_2014-08-12_RCAagain_20x_4k_Pos1_z00_ch00.tif
*#*Manufacture : Electron Microscopy Diatome --> 16% PARAFORMALDEHYDE AQ SOLUTN 50-980-486, No.:15700
*#Step# after confocal imaging
*#*Supply : (Fisher Scientific), 50980486
*#*Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF
*#*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
*#*Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
*#Take Tissue glass slide from -80C, and put them  to room temp. for 2min.
*#Maximum Intensity Projection(MIP) at each channel
*#Add 4% formamide on a tissue, incubate for 30min at room temp.
*#*Re-format the Step1~Step7 --> Step1~21
*#Wash with PBS 3 times (add PBS in 3 wells of 6 well plate and series washing with glasses)
*#*Step1 --> Step1:488, Step2:Cy3, Step3:Cy5
*#Attach glass with glue
*#*Step2 --> Step4:488, Step5:Cy3, Step6:Cy5
*#*Cyanoacrylate adhesives : High-viscosity, Sigma-Aldrich, Z105902-1EA
*#*...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5
*#Wipe PBS from the glass (around tissue)
*#Image aligning
*#Apply glue along the edge of the glass, put on the bottom of MatTek dish, wait for 5min
*#*Step# in one cycle is an exact position, no shift happened --> Cycle N = Step[3(N-1)+1], Step[3(N-1)+2], Step[3(N-1)+3]
[[File:TissueGlassOnMatTek_2.png|550px]]
*#*Get offset using Bright field images (ch03) of each Cycle : Step1, Step4, Step7, ... Step19
[[File:TissueGlassOnMatTek_1.png|400px]]
*#**I've done by manually, but eventually need automation
*#Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
*#*Name : Crop_[file name]
*#*ex)Offset
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Cycle'''
| align="center" style="background:#f0f0f0;"|'''X'''
| align="center" style="background:#f0f0f0;"|'''Y'''
|-
| align="center" |  1 || align="center" | 0  || align="center" | 0
|-
| align="center" | 2 || align="center" | -13 || align="center" | +8
|-
| align="center" | 3 || align="center" | -9  || align="center" | +8
|-
| align="center" | 4 || align="center" | -19 || align="center" | +4
|-
| align="center" | 5 || align="center" | -16  || align="center" | +4
|-
| align="center" | 5 || align="center" | -15  || align="center" | +10
|-
| align="center" | 5 || align="center" | -6  || align="center" | -9
|-
| align="center" | Average Offset || align="center" | -11.14  || align="center" | +0.71
|-
| align="center" | Round || align="center" | -11  || align="center" | +1
|-
| align="center" | New Offset || align="center" |  || align="center" |
|-
| align="center" |  1 || align="center" | -11  || align="center" | +1
|-
| align="center" | 2 || align="center" | -2 || align="center" | +7
|-
| align="center" | 3 || align="center" | +2  || align="center" | +7
|-
| align="center" | 4 || align="center" | -8 || align="center" | +3
|-
| align="center" | 5 || align="center" | -5  || align="center" | +3
|-
| align="center" | 5 || align="center" | -4  || align="center" | -11
|-
| align="center" | 5 || align="center" | +5  || align="center" | -10
|-
| align="center" | Crop Size || align="center" | 12  || align="center" | 12
|}


=====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.


*2. Run Script for detected barcodes and counts(RolonyDataInfo_v12_7StageDecoding.m, Rolony_Decoding_v18c_7StageDecoding.m)
*#Run PISA with every image (Step1~21)
*#*PISA parameter
*#*PISAParam.log_radius = 2;
*#*PISAParam.log_upper = -2e-4;           
*#*PISAParam.area_upper = 100;           
*#*PISAParam.area_lower = 10;           
*#*PISAParam.axratio_lower = 0.5;           
*#*PISAParam.circ_upper = 1.6;           
*#*PISAParam.circ_lower = 0.8;           
*#*PISAParam.pconn = 8;           
*#*PISAParam.bkgmult_lower = 3;           
*#*PISAParam.FilterOption = 1;       
*#*PISAParam.Deconvoluted = 0;
*#*PISAParam.DoMedflt = 1;
*#*PISAParam.nofindbkg = 0;
*#*eventually masking each image with MIP image will be used
*#Result gemerated : Barcodes, Counts


*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


*3. Mapping detected barcode string to gene
*#Matt made master key table : barcode and gene name
*#Allow 1 bit error (--> but these barcodes of 1 bit error were found in other genes...)
*#Map detected barcode to gene name using VLOOKUP(E#, Sheet0Ex!$A$2:$B$24000, 2, 0)


*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
====Result====
*#Wash with PBS once
*Saved at 2014-08-18
*#add 200uL BS(PEG)9 mix (4uL BS(PEG)9 in 196uL 1x PBS), incubate for 1hr at RT
**Total num : 2471
*#Wash with PBS once, add Tris pH 8.0, incubate for 30min
**Num detected only one dye at each digit : 1100
*#Wash with PBS once
**Num detected 488 and Cy3 at each digit : 775
**Num detected 488 and Cy5 at each digit : 785
**Num detected Cy3 and Cy5 at each digit : 275
**Num detected 488, Cy3 and Cy5 at each digit : 99


*Mapped to gene : 348
*Need to consider features with two or three dyes to be mapped to gene --> need more to polish the data


*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


 
[[File:DecodeResultSummary_2014-08-18.png|700px]]
 
=====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

Latest revision as of 22:24, 22 August 2014


Decoding with Agi26k0gap Padlock Probe[edit]

  • Image resolution : 20x obj, 4096 x 4086, z step = 1.04um
    • Since how many features would be detected or how much dense rolonies in this sample, so image in wide field of view
    • eventually 63x obj.
  • Data is in '2014-08-13'


Analysis[edit]

  • 1. Arrange data
    1. File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif
      • ex) PGP1F : PGP1F_Agi26k0gap_Decode_P20_S2_2014-08-09_20x_4k_Step1_Pos2_z01_ch01.tif
      • ex) Tissue section : HBTissue_2014-08-12_RCAagain_20x_4k_Pos1_z00_ch00.tif
    2. Step# after confocal imaging
      • Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF
      • Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
    3. Maximum Intensity Projection(MIP) at each channel
      • Re-format the Step1~Step7 --> Step1~21
      • Step1 --> Step1:488, Step2:Cy3, Step3:Cy5
      • Step2 --> Step4:488, Step5:Cy3, Step6:Cy5
      • ...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5
    4. Image aligning
      • Step# in one cycle is an exact position, no shift happened --> Cycle N = Step[3(N-1)+1], Step[3(N-1)+2], Step[3(N-1)+3]
      • Get offset using Bright field images (ch03) of each Cycle : Step1, Step4, Step7, ... Step19
        • I've done by manually, but eventually need automation
    5. Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
      • Name : Crop_[file name]
      • ex)Offset
Cycle X Y
1 0 0
2 -13 +8
3 -9 +8
4 -19 +4
5 -16 +4
5 -15 +10
5 -6 -9
Average Offset -11.14 +0.71
Round -11 +1
New Offset
1 -11 +1
2 -2 +7
3 +2 +7
4 -8 +3
5 -5 +3
5 -4 -11
5 +5 -10
Crop Size 12 12


  • 2. Run Script for detected barcodes and counts(RolonyDataInfo_v12_7StageDecoding.m, Rolony_Decoding_v18c_7StageDecoding.m)
    1. Run PISA with every image (Step1~21)
      • PISA parameter
      • PISAParam.log_radius = 2;
      • PISAParam.log_upper = -2e-4;
      • PISAParam.area_upper = 100;
      • PISAParam.area_lower = 10;
      • PISAParam.axratio_lower = 0.5;
      • PISAParam.circ_upper = 1.6;
      • PISAParam.circ_lower = 0.8;
      • PISAParam.pconn = 8;
      • PISAParam.bkgmult_lower = 3;
      • PISAParam.FilterOption = 1;
      • PISAParam.Deconvoluted = 0;
      • PISAParam.DoMedflt = 1;
      • PISAParam.nofindbkg = 0;
      • eventually masking each image with MIP image will be used
    2. Result gemerated : Barcodes, Counts


  • 3. Mapping detected barcode string to gene
    1. Matt made master key table : barcode and gene name
    2. Allow 1 bit error (--> but these barcodes of 1 bit error were found in other genes...)
    3. Map detected barcode to gene name using VLOOKUP(E#, Sheet0Ex!$A$2:$B$24000, 2, 0)


Result[edit]

  • Saved at 2014-08-18
    • Total num : 2471
    • Num detected only one dye at each digit : 1100
    • Num detected 488 and Cy3 at each digit : 775
    • Num detected 488 and Cy5 at each digit : 785
    • Num detected Cy3 and Cy5 at each digit : 275
    • Num detected 488, Cy3 and Cy5 at each digit : 99
  • Mapped to gene : 348
  • Need to consider features with two or three dyes to be mapped to gene --> need more to polish the data


File:DecodeResultSummary 2014-08-18.png