Matt:LabNotes/2017-1-23: Difference between revisions

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#Add RCA mix and incubate at 30C overnight (~15hrs)
#Add RCA mix and incubate at 30C overnight (~15hrs)
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===Day 3===
===Day 3===
#Wash with 1X PBS once
#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
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
#Wash with 1X PBS twice
#Wash with 1X PBS twice
#Add 1M Tris pH 8.0 and incubate at RT for 30min
#Add 1M Tris pH 8.0 and incubate at RT for 30min
#Wash with 1X PBS twice
#Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
#Wash sample with 10mM HEPES
#Add beads to sample and incubate 5min at RT
#Wash sample with 10mM HEPES twice
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
#Wash with 1X PBS twice
#Add 1X Tris pH 8.0 for 30min at RT
#Wash with 1X PBS twice
#Wash with 1X PBS twice



Revision as of 20:11, 26 January 2017

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

  1. ppMALAT1_dc1 dc1-488 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG G TCTTGCGTGCGATACGGAGT A AATGGAGGTATGACATATAATCT
  2. ppMALAT1 dc2-Cy3 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG T CTACTTCGTCGCGTCAGACC A AATGGAGGTATGACATATAATCT
  3. ppACTB dc0-Cy3 /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATCCGACGG A CGTATCGGTAGTCGCAACGC AGGCAAAGGCGAGGCT
  4. 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

  1. Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Made 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out brain section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Skip pepsin step usually done here
  9. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  10. Add 50ul gel casting mix filtered and degassed
  11. Seal in plastic bag and vacuum out air before filling with argon
  12. Let sit at RT for 30min
  13. Attach coverslip to bottom of petri dish
  14. Aspirate non-polymerized gel and wash twice with 1X PBS
  15. 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
  1. 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

  1. Wash with 1X PBS preheated to 55C with sample on 55C hot plate
    • Wash 2x for sample #1
    • Wash 10x for sample #2
  2. Add SplintR Enzyme and incubate at 37C for 20min
    • 20ul 10X Buffer + 6ul SplintR + 174ul H2O
  3. Wash with 1X PBS twice
  4. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
  5. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  6. 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
  1. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 3

  1. Wash with 1X PBS twice (normally once)
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice

Imaging

  1. Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice

Imaged on 12-5-2016-->