Arichard:Notebook/2013/June: Difference between revisions

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>Andrew
>Andrew
Line 52: Line 52:
** 2.5 ul 1:1e4 ERCC
** 2.5 ul 1:1e4 ERCC
** 14 ul H2O
** 14 ul H2O
* Total = 19 ul
** Total = 19 ul


* RT annealing mix:
* RT annealing mix:
Line 60: Line 60:
** 2 ul RNasin Plus
** 2 ul RNasin Plus
** 19 ul UHRR/ERCC mix
** 19 ul UHRR/ERCC mix
* Total = 30 ul
** Total = 30 ul


* RT rxn mix:
* RT rxn mix:
Line 67: Line 67:
** 2.5 ul 0.1 M DTT
** 2.5 ul 0.1 M DTT
** 2.5 ul SuperScript III
** 2.5 ul SuperScript III
* Total = 20 ul
** Total = 20 ul


* RT annealing rxn:
* RT annealing rxn:
** 12 ul RT priming buffer (Quartz seq adds this to beads, but we are using purified reference RNA in the priming buffer)
** 12 ul RT priming buffer (Quartz seq adds this to beads, but we are using purified reference RNA in the priming buffer)
* Total = 12 ul
** Total = 12 ul
# 1 min @ RT
# 1 min @ RT
# 90 sec @ 70 degC
# 90 sec @ 70 degC
Line 81: Line 81:
** 12 ul RT annealing rxn
** 12 ul RT annealing rxn
** 8 ul RT rxn mix
** 8 ul RT rxn mix
* Total = 20 ul
** Total = 20 ul
# 5 min @ 35 degC
# 5 min @ 35 degC
# 20 min @ 45 degC
# 20 min @ 45 degC
Line 95: Line 95:
** 3 ul Exo I
** 3 ul Exo I
** 23 ul H2O
** 23 ul H2O
* Total = 30 ul
** Total = 30 ul


* RT primer removal:
* RT primer removal:
** 20 ul RT rxn
** 20 ul RT rxn
** 36 ul Ampure RNAClean XP
** 36 ul Ampure RNAClean XP
* Total = 56 ul
** Total = 56 ul
# 10 min @ RT
# 10 min @ RT
# 5 min on magnet
# 5 min on magnet
Line 120: Line 120:
** 0.84 ul TdT (Roche)
** 0.84 ul TdT (Roche)
** 5.46 ul H2O
** 5.46 ul H2O
* Total = 10 ul
** Total = 10 ul


* PolyA rxn:
* PolyA rxn:
** 6 ul Exo I rxn
** 6 ul Exo I rxn
** 5 ul PolyA mix
** 5 ul PolyA mix
* Total = 11 ul
** Total = 11 ul
# 50 sec @ 37 degC
# 50 sec @ 37 degC
# 10 min @ 65 degC
# 10 min @ 65 degC
Line 136: Line 136:
** 6.25 ul Terra polymerase
** 6.25 ul Terra polymerase
** 58.75 ul H2O
** 58.75 ul H2O
* Total = 144.25 ul
** Total = 144.25 ul


* 2nd strand rxn:
* 2nd strand rxn:
** 11 ul PolyA rxn
** 11 ul PolyA rxn
** 46 ul 2nd strand mix
** 46 ul 2nd strand mix
* Total = 57 ul
** Total = 57 ul
# 10 sec @ 98 degC
# 10 sec @ 98 degC
# 1 min @ 40 degC
# 1 min @ 40 degC
Line 153: Line 153:
** 1 ul 100 uM SuppressPCR primer
** 1 ul 100 uM SuppressPCR primer
** 25.25 ul H2O
** 25.25 ul H2O
* Total = 52.33 ul
** Total = 52.33 ul


* PCR mix (KAPA):
* PCR mix (KAPA):
Line 159: Line 159:
** 1 ul 100 uM SuppressPCR primer
** 1 ul 100 uM SuppressPCR primer
** 25.25 ul H2O
** 25.25 ul H2O
* Total = 52.33 ul
** Total = 52.33 ul


* PCR rxn:
* PCR rxn:
** 57 ul 2nd strand rxn
** 57 ul 2nd strand rxn
** 50 ul PCR mix
** 50 ul PCR mix
* Total = 107 ul
** Total = 107 ul


* Clontech protocol:
* Clontech protocol:
Line 184: Line 184:
# Hold @ 4 degC
# Hold @ 4 degC
----------------------------------
----------------------------------
* Store @ -80 degC until library prep  
* Store @ -80 degC until library prep


===June 26, 2013===
===June 26, 2013===

Revision as of 21:14, 28 June 2013

June 2013

June 2, 2013

File:2013 06 02 hela5before.tif Cell 5, HeLa, before expelling onto glass top tube cap

File:2013 06 02 hela5after.tif Cell 5, HeLa, after expelling. The cell here is in the lower left of center. The other two objects are appear small, irregular, and shriveled as the focal plane is moved through them. This is difficult to convey in a single image.

File:2013 06 02 hela6before.tif Cell 6, HeLa, before expelling

File:2013 06 02 hela6after.tif Cell 6, HeLa, after expelling. There legitimately appears to multiple cells in this droplet.

  • These samples were diluted to ~10,000 cells/ml --> 10 cells/ul. More dilution might help avoid picking multiple cells, but other technical challenges remain.
  • After depositing the cell on the glass top tube cap, there is only ~10 seconds before the droplet dries (~100 nl maximum volume by my estimate, although variance is large.) This is hardly enough time to image the cell.
  • The CEL-Seq paper refers to "pipetting off excess liquid." I haven't tried this yet. I could deposit the cell in a larger volume, and then adjust the volume down. This issue here would be finding the cell in that larger volume (if I fail to see it shoot out of the pipette.) Also, the omnipresent dust particle and cell debris make imaging the cells very hard in BF.
  • I should probably wash the cells after thawing.

June 5, 2013

June 5, 2013 progress report

June 25, 2013

  • Implemented Quartz seq, Samples A1 and A2.
    • Original Quartz-seq protocols:

http://bit.accc.riken.jp/protocols/

    • Sample A1: 500 pg UHRR, CEL-Seq equivalent ERCC, Quartz purified RNA protocol. Used 2x Terra Direct PCR Mix.
    • Sample A2: Same, except I used 2x KAPA HiFi Uracil+.
  • Both samples had 1 ul of 1:500 dilution of 100 mM dUTP during final PCR (107 ul total volume).

Quartz-Seq Protocol for 500 pg Agilent UHRR, 2 samples

  • Work in the PCR hood
  • Keep enzyme stocks in cold box
  • Mix reactions on cold rack
  • Use UV treated Ambion RNase/DNase free water
  • Use UV treated 0.5 and 1.5 ml Eppendorf Lo-Bind tubes
  • Use UV treated 0.2 ml Axygen Cat# 22-154LR low retention tubes

  • UHRR/ERCC mix:
    • 2.5 ul 500 pg/ul UHRR
    • 2.5 ul 1:1e4 ERCC
    • 14 ul H2O
    • Total = 19 ul
  • RT annealing mix:
    • 3 ul 10X Titanium Taq buffer
    • 3 ul 1 mM dNTPs
    • 3 ul 0.833 uM RT primer
    • 2 ul RNasin Plus
    • 19 ul UHRR/ERCC mix
    • Total = 30 ul
  • RT rxn mix:
    • 2 ul 10X Titanium Taq buffer
    • 13 ul H2O
    • 2.5 ul 0.1 M DTT
    • 2.5 ul SuperScript III
    • Total = 20 ul
  • RT annealing rxn:
    • 12 ul RT priming buffer (Quartz seq adds this to beads, but we are using purified reference RNA in the priming buffer)
    • Total = 12 ul
  1. 1 min @ RT
  2. 90 sec @ 70 degC
  3. 15 sec @ 35 degC
  4. Hold @ 4 degC

  • RT rxn:
    • 12 ul RT annealing rxn
    • 8 ul RT rxn mix
    • Total = 20 ul
  1. 5 min @ 35 degC
  2. 20 min @ 45 degC
  3. 10 min @ 70 degC
  4. Hold at 4 degC
  5. Prepare Exo I mix

  • Exo I mix:
    • 2 ul 10X Titanium Taq buffer
    • 1 ul 10X Exo III buffer
    • 1 ul 0.1 M DTT
    • 3 ul Exo I
    • 23 ul H2O
    • Total = 30 ul
  • RT primer removal:
    • 20 ul RT rxn
    • 36 ul Ampure RNAClean XP
    • Total = 56 ul
  1. 10 min @ RT
  2. 5 min on magnet
  3. Wash 2x with 1 min with 50 ul 80% EtOH
  4. Dry 3 min
  5. Add 6 ul Exo I mix
  6. 1 min @ RT
  7. 5 min on magnet
  8. Transfer to new tube
  9. 30 min @ 37 degC
  10. 20 min @ 80 degC
  11. Hold @ 4 degC

  • PolyA mix:
    • 1 ul 10X Titanium Taq buffer
    • 1.5 ul 20 mM dATP (dilute 100 mM dATP 1:5)
    • 1.2 ul 1:5 RNase H
    • 0.84 ul TdT (Roche)
    • 5.46 ul H2O
    • Total = 10 ul
  • PolyA rxn:
    • 6 ul Exo I rxn
    • 5 ul PolyA mix
    • Total = 11 ul
  1. 50 sec @ 37 degC
  2. 10 min @ 65 degC
  3. Hold @ 4 degC

  • 2nd strand mix:
    • 78.25 ul 2x Terra buffer
    • 1 ul 10 uM Tagging primer
    • 6.25 ul Terra polymerase
    • 58.75 ul H2O
    • Total = 144.25 ul
  • 2nd strand rxn:
    • 11 ul PolyA rxn
    • 46 ul 2nd strand mix
    • Total = 57 ul
  1. 10 sec @ 98 degC
  2. 1 min @ 40 degC
  3. 5 min @ 68 degC
  4. Hold @ 4 degC
  5. Move to cold rack

  • PCR mix (Clontech):
    • 21.1 ul 2x Terra
    • 1 ul 100 uM SuppressPCR primer
    • 25.25 ul H2O
    • Total = 52.33 ul
  • PCR mix (KAPA):
    • 21.1 ul 2x HiFi U+
    • 1 ul 100 uM SuppressPCR primer
    • 25.25 ul H2O
    • Total = 52.33 ul
  • PCR rxn:
    • 57 ul 2nd strand rxn
    • 50 ul PCR mix
    • Total = 107 ul
  • Clontech protocol:
  1. Preheat, 10 sec @ 68 deg
  2. 15 cycles:
    1. 10 sec @ 98 degC
    2. 15 sec @ 65 degC
    3. 5 min @ 68 degC
  3. 5 min @ 68 degC
  4. Hold @ 4 degC
  • KAPA protocol
  1. 5 min @ 95 degC
  2. 15 cycles:
    1. 20 sec @ 98 degC
    2. 15 sec @ 65 degC
    3. 5 min @ 72 degC
  3. 5 min @ 72 degC
  4. Hold @ 4 degC

  • Store @ -80 degC until library prep

June 26, 2013

  • Library prep for Quartz-seq samples A1 and A2:
    • Adapter ligation using KAPA Rapid Ligation, USER, and PCR with KAPA SYBR Fast (See Jeff and Noi's protocols).

CORE fragmentation and library prep

  • Work in PCR hood
  • UV treat H2O, tips, tubes
  • Purified Quartz WTA products using Ampure XP beads
    • Added 100 ul beads to 107 ul product
    • Eluted in 14.2 ul H2O
  • UDG/Endo IV rxn
    • 14.2 ul template
    • 1 ul 1:121 UDG
    • 1 ul 1:121 Endo IV
    • 1.8 ul Exo- buffer
  • Total = 18 ul
  1. 2 hrs @ 37 degC
  2. 15 min @ 65 degC
  3. Hold @ 4 degc

  • Nick translation
    • 18 ul UDG/Endo IV rxn
    • 1 ul 1:50 Exo-
    • 1 ul 1:25 1 mM dNTP (40 uM)
  • Total = 20 ul
  1. 1 hrs @ 37 degC
  2. 15 min @ 75 degC
  3. Hold @ 4 degC

  • Ligation rxn
    • 20 ul nick translation rxn
    • 25 ul 2X KAPA ligation buffer
    • 1.67 ul 1.5 uM loop adapter
    • 1.33 ul H2O
    • KAPA DNA ligase
  • Total = 50 ul
  1. 15 min @ 20 degC
  2. Add 2 ul USER
  3. 15 min @ 37 degC
  4. Hold @ 4 degC

  • Ampure XP bead purification
    • 50 ul beads
    • Elute in 20 ul H2O
  • PCR
    • 20 ul template
    • 1 ul 10 uM PCR_F
    • 1 ul 10 uM NEBNext index
    • 3 ul H2O
    • 2X KAPA SYBR mix
  • Total = 50 ul
  1. 30 sec @ 98 degC
  2. 15 cycles:
    1. 10 sec @ 98 degC
    2. 30 sec @ 65 degC
    3. 45 sec @ 72 degC
  3. 2 min @ 72 degC
  4. Hold @ 4 degC

File:2013 06 26 quartz seq 500 pg uhrr.jpd

June 27, 2013