Matt:LabNotes/2015-9-29: Difference between revisions
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#*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C | #*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 | #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 | #To DARTFISH slide: Add 100nM Padlock Probe Mix with 1000X suppv2 oligos in one sample and only 100nM Padlock Probe Mix in other 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 | #*Put in pipette tip box with bottom filled with water and parafilmed around edges of box | ||
{| {{table}} | {| {{table}} | ||
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|- | |- | ||
| 100nM suppv2 Oligos || 28.57 | | 100nM suppv2 Oligos || 28.57 | ||
|- | |||
| Ampligase||10 | |||
|- | |||
| Total||100 | |||
|} | |||
{| {{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 | |||
|- | |- | ||
| Ampligase||10 | | Ampligase||10 |
Revision as of 01:28, 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)
- Two sections per glass slide
- Do one FISSEQ and one DARTFISH in culture dish as control
- DARTFISH coverslip glass cracked
Protocol
Day 1
- Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Made 40ml 4% PFA at RT
- 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
- Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- 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 gaskets to glass slides
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Let sit in PBS for ~10min while preparing RT Mix
- 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 |
- 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 in one sample and only 100nM Padlock Probe Mix in other 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 |
Component | Volume |
DEPC-H2O | 20.18 |
Ampligase Buffer | 10 |
Padlock Probes ___nM | 31.25 |
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
Component | 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
Component | 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 1:2000 diluted beads for 3 minutes in 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