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

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*Derek bead purified Second Exp sample for sequencing
*Derek bead purified Second Exp sample for sequencing
**Labeled as 2015 05 29 MC E and quantified
**Labeled as 2015 05 29 MC E and quantified
[[File:2015-06-01_BA8SmartSeq_BeadPurify_GelCheck.jpg | 250px]]
*I bead purified Second PosCtrl sample for sequencing
**Labed as 2015 06 16 MC POSCTRL and quantified
[[File:2015-06-01_BA8SmartSeq_BeadPurify_GelCheck.jpg | 250px]] [[File:2015-06-16_UHRRPosCtrlSMARTSeq_BeadPurify_GelCheck.jpg | 200px]]

Latest revision as of 23:46, 16 June 2015

SMART-Seq of BA8 Tissue Section[edit]

Purpose[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • Make Master Mix
    • Add polymerase just before use (and don't vortex)
Component Volume
2X SeqAmp PCR Buffer 82.5
PCR Primer II A v3 (12uM) 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 9 -> 72C 10min -> 4C forever

  • Ampure XP bead purification with 50ul beads, elute 17ul with H2O
  • 1ul for Qubit HS dsDNA:
    • PosCtrl: 2.53 ng/ul
    • Exp: 0.452 ng/ul
    • NegCtrl: out of range

Nextera XT Tagmentation[edit]

  • Input is ~1ng
  1. Add 4 ul 5X Tn5 buffer to each PCR tube
  2. Transfer 14ul of each sample to tubes
    • PosCtrl: 0.4ul sample + 13.6ul H2O
    • Exp: 2.2ul sample + 11.8ul H2O
    • NegCtrl: 14ul sample
  3. Add 2 ul Tn5 (1:50 dilution in 1:1 TE:glycerol) to each sample. Incubated 5 minutes at 55C
    • Old Tn5 from Epicentre
  4. Add 1ul Qiagen protease (20mg/ml) to each sample and incubate 15 minutes at 50C, followed by 20 minutes at 70C

Adapter PCR[edit]

  1. Make PCR master mix (per reaction):
    1. 10 ul 3.3x NPM
    2. 0.66 ul S511 Adapter
    3. 1.65 ul SYBR Green (10X, final needs to be 0.5X)
  2. Add 21ul of Nextera sample
  3. Add 0.66 of N7XX barcode adapters to each sample
    1. PosCtrl:N701
    2. Exp:N702
    3. NegCtrl:N703
  4. Add 11.3 ul of PCR master mix to each sample
  5. Incubate in thermocycler:
    1. 72C for 3min
    2. 95C for 30sec
    3. 12 cycles of:
      1. 95C for 10sec
      2. 55C for 30sec
      3. 72C for 30sec
    4. 72C for 5min
  • qPCR shows no increase in fluorescence after 12 cycles

Nextera XT[edit]

  • Repeat with Derek's/Blue's reagents and protocol
  • Input ~0.2ng DNA
  • PosCtrl: 0.253ng/ul (diluted 1:10)
  • Exp: 0.226ng/ul (diluted 1:2)
  • NegCtrl: H2O
  • GM12878 gDNA: 0.201ng/ul (diluated 0.803 ng/ul 1:4)
  1. Add 2.5ul Tagment DNA Buffer
  2. Add 1.25ul Amplification Tagment Mix
  3. Add 1.25ul Sample
  4. Vortex and spin down
  5. Incubate 55C for 10min and then hold at 10C
  6. Add 1.25ul NT buffer and incubate at RT for >5min
  1. Add 3.75ul NPM, 1.25ul Index Primer 1 and 1.25ul Index Primer 2 (S517)
    • PosCtrl: N701
    • Exp: N702
    • NegCtrl: N703
    • gDNA: N704
  2. Vortex and spin down
  1. Incubate in thermocycler:
    1. 72C for 3min
    2. 95C for 30sec
    3. 12 cycles of:
      1. 95C for 10sec
      2. 55C for 30sec
      3. 72C for 60sec
    4. 72C for 5min

Gel Check[edit]

  1. Run 1 ul of each sample on PAGE gel with 2ul H2O 3 ul 6X loading dye
    1. 0.5 ul Low mass Ladder
    2. 25 min @ 250 V
  • Lane1:First PosCtrl
  • Lane2:First Exp
  • Lane3:First NegCtrl
  • Lane4:Low Mass Ladder
  • Lane5:Second PosCtrl
  • Lane6:Second Exp
  • Lane7:Second NegCtrl
  • Lane8:Second gDNA

File:2015-05-29 BA8 SMARTSeq Nextera GelCheck.jpg

  • Looks like Nextera with 1ng input and Andrew's reagents (SYBR Green) did not work
  • Following recommended protocol of 0.1-0.3ng input and Derek/Blue's reagents did work!
  • Derek bead purified Second Exp sample for sequencing
    • Labeled as 2015 05 29 MC E and quantified
  • I bead purified Second PosCtrl sample for sequencing
    • Labed as 2015 06 16 MC POSCTRL and quantified

File:2015-06-01 BA8SmartSeq BeadPurify GelCheck.jpg File:2015-06-16 UHRRPosCtrlSMARTSeq BeadPurify GelCheck.jpg