Matt:LabNotes/2015-3-31

From ZhangLabWiki
Jump to navigation Jump to search

Sequence in situ Rolonies via in situ Slide PCR[edit]

  • Purpose: Sequence the rolonies in situ to quantify the contributions of each probe to the decoded rolonies
    • ie gene GNG4 has many rolonies but are they due to a single highly efficient probe or a number of different probes

Protocol[edit]

  • Samples:
    • Exp: BA8 CA12k_Nov2014_V4 Captured Rolonies
    • NegCtrl: BA8 1st (cDNA) Rolonies (from old FISSEQ protocol)

Strip hybridized dye-probes with 80% formamide pre-heated to 80C[edit]

  • The tissue is on thin cover-slip placed on a glass slide
  • Take cover-slip off glass slide and put in small culture dish
  • Incubate for 15min at RT and then wash twice with 1X PBS

File:033115 SeqRolony Strip.JPG

Slide PCR[edit]

  • Make PCR Reaction Mix on ice
Components Volume 2X Volume
100uM SeqRolony_CL_Amplify 1 2
2X Taq Master Mix 50 100
H2O 49 98
Total 100 200
  • Place cover-slip on clean glass slide (tissue face up)
  • Use silicon isolator with circular opening to seal tissue
    • NegCtrl silicon had large gaps so used glue to glue all around the circle on bottom of silicon

File:033115 SeqRolony NegCtrl.JPG

    • Exp had very small leak so used glue to seal the edges of silicon

File:033115 SeqRolony ExpBefore.JPG

  • Add 100ul PCR mix and seal with plastic to prevent evaporation

File:033115 SeqRolony ExpAfter.JPG

  • Put on thermocycler with slide adapter
 Program 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold
  • Remove plastic seal and pipette solution into PCR tubes

File:033115 SeqRolony ExpExtract.JPG

    • NegCtrl had ~90ul (good) but Exp was completely dry (bad, probably leaked out as evidenced by white haze on glass slide outside of silicon)
  • Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon)
  • Final amount: 190ul NegCtrl and 100ul Exp

Add Sequencing Adapters[edit]

  • Qiagen PCR purification
    • Elute 40ul
  • Nanodrop ssDNA:
    • Exp: 0.6 ng/ul
    • NegCtrl: 2.2 ng/ul

PCR Test[edit]

Components 1X Volume 3X Volume
Slide PCR amplicon 11.7 0
SeqRolony_P5 0.4 1.2
SeqRolony_P7 0.4 1.2
2X KAPA SYBG MM 12.5 37.5
Total 25 60
  • Aliquot 13.3ul from 3X master mix and add 11.7ul slide amplicon
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

File:033115 SeqRolony PCRTest.JPG

Gel Check[edit]

  • TBE Gel with 4ul sample + 4ul 6X Loading Dye
    • 0.5ul Low Mass Ladder

File:2015-04-01 SeqRolony PCRtest.jpg

  • Main bright band is the correct size (~225bp <- 150bp padlock probe + 75bp adapters)

PCR[edit]

Components 1X Volume 2.5X MM
Slide PCR amplicon 27 0
SeqRolony_P5 1 2.5
SeqRolony_P7 1 2.5
2X KAPA SYBG MM 50 125
H2O 21 52.5
Total 100 187.5
  • Aliquot 73ul from 2.5X master mix and add 27ul captured template
 Program
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x9 -> 72C 3min

File:040115 SeqRolony PCR.JPG

  • Qiagen PCR Purification
    • Elute 40ul
  • Nanodrop measure concentration
    • Exp: 45.5 ng/ul * 38ul = 1.729ug
    • NegCtrl: 27.9 ng/ul * 38ul = 1.060ug

Size Selection[edit]

  • 5-well TBE Gel 220V for 32min
    • Low mass ladder
    • 3 lanes of Exp
    • 1 lane of NegCtrl
  • Cut out the bright band at 225bp

File:2015-04-02 SeqRolony SizeSelect.jpg

  • Add to 2 0.5mls tube (with hole at bottom) in 2ml tubes
    • Spin at 15,000rpm for 3 min
  • Add 450ul 1X TE Buffer to each and shake at 37C for 1 hour
  • Filter out gel with 2 Nanosep columns
    • Spin at 15,000rpm for 3 min
    • Transfer solution, spin, then transfer gel and spin
  • Add 1000ul 100% EtOH, 45ul 3M NaOAc, and 1.4ul glycoblue
    • Vortex
  • Put in -80C freezer for 30min
  • Spun at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 15ul and combine
  • FINAL: 30ul in 1.5ml tube labeled "MC-SeqRolony_BA8V4_Mar3115-13" and put in Non-quantified New Sequencing Libraries box with SeqRolony_RP1 and SeqRolony_IRP