Matt:LabNotes/2017-1-23
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Experiment: Increase # of wash cycles between hybridization and ligation to improve specificity
Background
- DART-FISH with SplintR using probe set with unused barcodes and stained with Anti-NeuN after revealed
- high number of decoded rolonies with unused barcodes
- many glia-specific rolonies in cells stained with anti-NeuN
- this is very problematic for using rolony gene markers as a way to classify cell types
- probably due to low specificity (padlock probe hybridization and/or ligation)
- hybridization is carried out at 55C for ~20hr
- sample was then washed twice with 55C PBS
- then hybridized probes were ligated at 37C by SplintR for 20min
- my hypothesis is that falsely ligated probes are due to incomplete removal of non hybridized probes during wash step
- by increasing washes should see decrease in false positives
Experimental Design
- Use IDT ordered padlock probes to capture RNA of housekeeping genes and neuron-specific genes (RELN)
- Add all probes to 3 samples
- Each sample is washed different number of times (2, 6, 15)
- Rolonies are imaged and then samples are stained with DRAQ5 and Anti-NeuN
- Check percentage of RELN not in Anti-NeuN stained cells
Padlock Probes
Housekeeping padlock probes
- ppMALAT1_dc1 dc1-488 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG G TCTTGCGTGCGATACGGAGT A AATGGAGGTATGACATATAATCT
- ppMALAT1 dc2-Cy3 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG T CTACTTCGTCGCGTCAGACC A AATGGAGGTATGACATATAATCT
- ppACTB dc0-Cy3 /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATCCGACGG A CGTATCGGTAGTCGCAACGC AGGCAAAGGCGAGGCT
- ppRAB7A dc1-488 /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATCCGACGG G TCTTGCGTGCGATACGGAGT AGAGGAGACTAAACGGAGGACA
RELN padlock probes
- Name dcProbe Probe T4count SplintRcount Ampligasecount
- ppRELN_1 dc5-Cy3+dc6-488 RELN_AAAAGAGGTTGTTTCCACTAGAAAGGATTCCACACTTTCGAGATGGTTTCC 43 67 123
- /5Phos/ ATTCCACACTTTCGAGATGGTTTCC CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG CACGCTTACGATCCCGCTAT AAAAGAGGTTGTTTCCACTAGAAAGG
- ppRELN_2 dc5-Cy3+dc6-Cy3 RELN_CTCTGTGATGCCTGAACACTTGTAGATGTGTATAGTCCTGTCACCAGCAA 19 31 70
- /5Phos/ ATGTGTATAGTCCTGTCACCAGCAA CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG TCGTAACCCGTGCGAAGTGC CTCTGTGATGCCTGAACACTTGTAG
- ppRELN_3 dc5-Cy3+dc6-Cy5 RELN_GAAAATTCCAGTCTCACTGGATCCGCGGATGAGCTATCAGTCGAACAGC 70 11 20
- /5Phos/ CGGATGAGCTATCAGTCGAACAGC CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG CTCTCGTAGCGTGCGATGAG GAAAATTCCAGTCTCACTGGATCCG
Protocol
- 3 Samples: GTEx Patient 5342 Occipital Cortex sectioned by Yun on 11-22-2016
- 5% Gel mix and protocol is same as this with Acryloyl-X
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
10mg/ml Acryloyl-X, SE in DMSO | 1 |
H2O | 39.75 |
5% TEMED | 1 |
5% APS | 1 |
Total | 50 |
Day 1
- Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Made 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out brain section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Skip pepsin step usually done here
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash twice with 1X PBS
- Prepare SplintR Mix
- 1ul of each 100uM probes (4) and 0.5ul of each 200uM probes (2) + 595ul H2O for 1uM total conc
- In SplintR Mix each probe has concentration = 100nM
Component | Volume |
10X SplintR Buffer | 10 |
Padlock Probe Mix 1uM | 60 |
H2O | 30 |
Total | 100 |
- Move sample to 60C HybEZ oven and slowly decreases to 55C and held for ~2hr
- Only 2 samples because 1 cracked
- The 2 samples were left at 4C in 1X PBS for 1 or 2 hrs before adding SplintR Mix
Day 2
- Wash with 1X PBS preheated to 55C with sample on 55C hot plate
- Wash 2x for sample #1
- Wash 10x for sample #2
- Add SplintR Enzyme and incubate at 37C for 20min
- 20ul 10X Buffer + 6ul SplintR + 174ul H2O
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Add RCA mix and incubate at 30C overnight (~15hrs)
Day 3
- Wash with 1X PBS twice (normally once)
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) 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
Imaging
Settings
- 20X objective
- 2048x2048 px
- 4x line average
- 3 positions in each dish
Rolony FISH
- Add 0.5uM dc5_Cy3 in 30% formamide + 2X SSC preheated to 75C (detects RELN)
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Find positions near layer 1 of cortex with RELN and image
- Strip twice with 15min incubation of 80% formamide preheated to 75C
- Wash with 1X PBS twice after each strip
- Image to confirm no signal
- Add
- Wash with 2X SSC twice
- Image same positions
- Strip twice with 15min incubation of 80% formamide preheated to 75C
- Wash with 1X PBS twice after each strip
Anti-NeuN Staining Procedure
- Rinse tissue with 1X PBS for 30min
- Incubate section for 30min in 1ml blocking buffer
- 50ul DKS + 250ul 10% BSA + 0.1ul TX-100 + 100ul 10X PBS + 600ul H2O
- Incubate section for 1hr in 1ml NeuN antibody solution
- 1ul rabbit anti-NeuN (Millipore MABN140) diluted in 999ul blocking buffer same as in step 2
- Wash 3x 5min in 1X PBS
- Stain with Donkey anti-Rabbit Alexa 488 for 1.5hr
- DxR488 stock was diluted 1:200 in blocking buffer without the TX-100
- Wash 3x 5min in 1X PBS
- 2nd wash use 5uM DRAQ5 in 1X PBS
- 3rd wash use 1X PBS + 0.1% Tween-20
- Add 150ul Vectashield mounting media with antifade and allow 10min before imaging
DRAQ5 Staining
- Incubate in 5uM DRAQ5 + 1X PBS for 15min and don't wash before imaging again