Matt:LabNotes/2015-3-31
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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
- Primer design and general method: Matt:LabNotes/2015-3-14
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
- 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