Matt:LabNotes/2015-9-29: Difference between revisions

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(Created page with "=FISSEQ and DARTFISH= *Prepare FISSEQ and DARTFISH samples from human brain tissue (BA8) for Harvard on glass slides **Two sections per glass slide ***FISSEQ slide just do dup...")
 
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***DARTFISH slide do one without suppressorv2 (tissue close to frosted edge) and one with supperssorv2 (far from frosted edge)
***DARTFISH slide do one without suppressorv2 (tissue close to frosted edge) and one with supperssorv2 (far from frosted edge)
*Do one FISSEQ and one DARTFISH in culture dish as control
*Do one FISSEQ and one DARTFISH in culture dish as control
**DARTFISH coverslip glass cracked


==Protocol==
==Protocol==
===Day 1===
#Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
#Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
#*EtOH sterilize and UV
#*EtOH sterilize and UV
Line 12: Line 14:
#*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
#*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
#Take out BA8 section (2 glass slides and 2 coverslips) from -80C and incubate on 50C hot plate for 3min
#Take out BA8 section (2 glass slides and 2 coverslips) from -80C and incubate on 50C hot plate for 3min
#*Attach gaskets to glass slides
#Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
#Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
#Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
#Submerge glass slides in 30ml 4%PFA in 50ml tube for 15min at 37C
#Wash with 2X SSPE by submerging
#Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
#Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
#Aspirate any liquid and attach gaskets to glass slides
#Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
#Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
#Wash with nuclease-free H2O three times
#Wash with nuclease-free H2O three times
Line 21: Line 24:
#*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
#*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
#Wash with nuclease-free PBS three times
#Wash with nuclease-free PBS three times
#Prepare 6X Reverse Transcription Mix '''on ice'''
#*Let sit in PBS for ~10min while preparing RT Mix
#Prepare 5X Reverse Transcription Mix '''on ice'''
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 32: Line 36:
| 25mM dNTP||2
| 25mM dNTP||2
|-
|-
| 2mM aa-dUTP||4
| 4mM aa-dUTP||2
|-
|-
| 100uM N9 or 100uM FISSEQ_RT||5
| 100uM N9 or 100uM FISSEQ_RT||5
Line 43: Line 47:
|}
|}
#Incubate 10min at 4C and then ~18hr at 37C
#Incubate 10min at 4C and then ~18hr at 37C
#*Put in pipette tip box with bottom filled with water and parafilmed around edges of box
===Day 2===
#Wash with 1X PBS once
#Add 200ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 1X PBS) and incubate 1hr at RT
#Wash with 1X PBS twice
#Add Tris pH8.0 and incubate 30min at RT
#Wash with 1X PBS twice
#RNA Removal
#*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
#Wash with H2O twice
#To DARTFISH slide: Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
#*Put in pipette tip box with bottom filled with water and parafilmed around edges of box
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| DEPC-H2O||20.18
|-
| Ampligase Buffer||10
|-
| [[Matt:JustinLabNotes/ | Padlock Probes ___nM]]||31.25
|-
| 100nM suppv2 Oligos || 28.57
|-
| Ampligase||10
|-
| Total||100
|}
#To FISSEQ slide and dish: Add CircLigaseII mix and incuabate 1hr at 60C
#*Put in pipette tip box with bottom filled with water and parafilmed around edges of box
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| DEPC-H2O||128
|-
| CircLigase Buffer 10X||20
|-
| MnCl2 50mM||10
|-
| Betaine 5M|| 40
|-
| CircLigase II (100U/ul)||2
|-
| Total||100
|}
#Wash FISSEQ samples with PBS and store in PBS at 4C overnight
===Day 3===
#Wash with 1X PBS once
#Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
#*Preheated to 70C before adding
#Wash with 2X SSC once, 1X SSC once, and 1X PBS once
#Add RCA mix and incubate 6hr at 30C
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| H2O||172
|-
| 10X Phi29 Buffer||20
|-
| 25mM dNTP||2
|-
| 2mM aa-dUTP||4
|-
| Phi 29 (low conc)||2
|-
| Total||200
|}
#Wash with 10mM HEPES
#Sonicate [[Matt:LabNotes/2015-8-13#Add_Beads | 1:2000 diluted beads]] for 3 minutes in [https://contenti.com/l-r-pc3-compact-ultrasonic-cleaner sonicator bath]
#Add 200ul to sample and incubate 5min at RT
#Wash with 10mM HEPES once
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr
#Wash with 1X PBS twice
#Add 1M Tris pH 8.0 and incubate at RT for 30min
#Wash with 1X PBS twice and store in 1X PBS at 4C overnight

Revision as of 01:27, 30 September 2015

FISSEQ and DARTFISH

  • Prepare FISSEQ and DARTFISH samples from human brain tissue (BA8) for Harvard on glass slides
    • Two sections per glass slide
      • FISSEQ slide just do duplicates
      • DARTFISH slide do one without suppressorv2 (tissue close to frosted edge) and one with supperssorv2 (far from frosted edge)
  • Do one FISSEQ and one DARTFISH in culture dish as control
    • DARTFISH coverslip glass cracked

Protocol

Day 1

  1. Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 section (2 glass slides and 2 coverslips) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Submerge glass slides in 30ml 4%PFA in 50ml tube for 15min at 37C
  6. Wash with 2X SSPE by submerging
  7. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  8. Aspirate any liquid and attach gaskets to glass slides
  9. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
  10. Wash with nuclease-free H2O three times
  11. Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
  12. Wash with nuclease-free PBS three times
    • Let sit in PBS for ~10min while preparing RT Mix
  13. Prepare 5X Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 or 100uM FISSEQ_RT 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box

Day 2

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add Tris pH8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
  7. Wash with H2O twice
  8. To DARTFISH slide: Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box
Component Volume
DEPC-H2O 20.18
Ampligase Buffer 10
Padlock Probes ___nM 31.25
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
  1. To FISSEQ slide and dish: Add CircLigaseII mix and incuabate 1hr at 60C
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box
Component Volume
DEPC-H2O 128
CircLigase Buffer 10X 20
MnCl2 50mM 10
Betaine 5M 40
CircLigase II (100U/ul) 2
Total 100
  1. Wash FISSEQ samples with PBS and store in PBS at 4C overnight

Day 3

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. Add RCA mix and incubate 6hr at 30C
Component Volume
H2O 172
10X Phi29 Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
Phi 29 (low conc) 2
Total 200
  1. Wash with 10mM HEPES
  2. Sonicate 1:2000 diluted beads for 3 minutes in sonicator bath
  3. Add 200ul to sample and incubate 5min at RT
  4. Wash with 10mM HEPES once
  5. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr
  6. Wash with 1X PBS twice
  7. Add 1M Tris pH 8.0 and incubate at RT for 30min
  8. Wash with 1X PBS twice and store in 1X PBS at 4C overnight