Matt:LabNotes/2015-5-26: Difference between revisions

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>Mzcai
>Mzcai
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| DTT (100mM)||1.65
| DTT (100mM)||1.65
|-
|-
| dNTP Mix (20mM)||3.3
| dNTP Mix (10mM, protocol says should be 20mM)||3.3
|-
|-
| SMARTer IIA Oligo (12uM)||3.3
| SMARTer IIA Oligo (12uM)||3.3
Line 65: Line 65:
*Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever
*Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever
*Keep at in 4C fridge overnight
*Keep at in 4C fridge overnight
===SMART-Seq v3 PCR===
===SMART-Seq v3 PCR===
*Make Master Mix
*Make Master Mix

Revision as of 02:29, 27 May 2015

SMART-Seq of BA8 Tissue Section

Purpose

  • Do on same tissue sections we do DARTFISH on to compare RNA-Seq to DARTFISH
    • This time sequence from WHOLE tissue section (approx 4cm^2) to validate protocol
    • In future cut out small tissue section (approx 1mm^2) to match same region we do DARTFISH on (and maybe another small tissue section on opposite end)
  • Hypothesis: DARTFISH counts will correlate better with the small cut out tissue section than the whole section
    • This will support that DARTFISH can show regional differences in gene expression

Isolate RNA from BA8 tissue section

  • Using ZR RNA MicroPrep kit from Zymo
  1. Wipe all surfaces, forceps, etc with EtOH and RNase Zap
  2. Scrape tissue off glass with a scalpel and put in 1.5ml tube
  3. Add 400ul RNA Lysis Buffer
    • Vortex 10sec and pipette with 1000p ~10x
  4. Centrifuge at 18,000rcf for 1min
  5. Transfer the 400ul to IIIC column and centrifuge at 8,000rcf for 30sec
  6. Add 320ul 100% EtOH (UV'd) to flow-trhough and mix
  7. Transfer 720ul to IC column and centrifuge at 18,000rcf for 1min
  8. Add 400ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec
  9. Add 30ul DNase I cocktail and incubate in 37C incubator for 15min
    • 1.5ul DNase I (2U/ul) + 3ul 10X Reaction Buffer + 25.5ul RNA Wash Buffer
    • Centrifuge at 18,000rcf for 30sec
  10. Add 400ul RNA Prep Buffer and centrifuge at 18,000rcf for 1min
  11. Add 800ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec and repeat with 400ul Wash Buffer
  12. Spin in emptied collection tube at 18,000rcf for 2min
  13. Add 10ul H2O, let stand for 1min, and then centrifuge at 10,000rcf for 30sec
  • Nanodrop Measurement: 16.5ng/ul
    • A260/280 = 1.75
    • A260/230 = 1.60

SMART-Seq v3 RT

  • Do work prior to PCR in PCR clean hood
  • Prepare 10X Reaction Buffer: 19ul 10X Lysis Buffer + 1ul RNase Inhibitor (avoid bubbles when mixing)
  • Add 1ul 10X Reaction Buffer to 1ul of sample and 8ul H2O
    • NegCtrl: Nuclease-free H2O
    • PosCtrl: UHRR (10ng/ul)
    • Exp: Isolated total RNA (16.5ng/ul)
  • Add 1ul 3' SMART-Seq CDS Primer II A to each
  • Incubate at 72C for 3min followed by ice until next step
  • Make Master Mix
Component Volume
5X First-Strand Buffer 13.2
DTT (100mM) 1.65
dNTP Mix (10mM, protocol says should be 20mM) 3.3
SMARTer IIA Oligo (12uM) 3.3
Rnase Inhibitor (40U/ul) 1.65
SMARTScribe Reverse Transcriptase (100U/ul) 6.6
Total 29.7
  • Just prior to adding MM add the 6.6ul SMARTScribe Reverse Transcriptase to MM and mix without vortexing
  • Add 9ul of MM to each tube and mix with pipette and then spin down
  • Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever
  • Keep at in 4C fridge overnight

SMART-Seq v3 PCR

  • Make Master Mix
    • Add polymerase just before use (and don't vortex)
Component Volume
2X SeqAmp PCR Buffer 82.5
PCR Primer II A 3.3
SeqAmp DNA Polymerase 3.3
Nuclease-Free H2O 9.9
Total 99
  • Add 30ul to each sample
  • Take out of PCR clean hood

Thermalcycle: 95C 1min -> (98C 10s -> 65C 30s -> 68C 3min) x __ -> 72C 10min -> 4C forever

SMART-Seq v4 PCR

  • Make Master Mix
    • Add polymerase just before use (and don't vortex)
Component Volume
2X SeqAmp PCR Buffer 82.5
PCR Primer II A 3.3
SeqAmp DNA Polymerase 3.3
Nuclease-Free H2O 9.9
Total 99
  • Add 30ul to each sample
  • Take out of PCR clean hood

Thermalcycle: 95C 1min -> (98C 10s -> 65C 30s -> 68C 3min) x __ -> 72C 10min -> 4C forever