Arichard:Notebook/2013/June: Difference between revisions

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>Andrew
>Andrew
Line 56: Line 56:


* RT annealing mix:
* RT annealing mix:
** 3 ul 10X Titanium Taq buffer
3 ul 10X Titanium Taq buffer
** 3 ul 1 mM dNTPs
3 ul 1 mM dNTPs
** 3 ul 0.833 uM RT primer
3 ul 0.833 uM RT primer
** 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:
** 2 ul 10X Titanium Taq buffer
2 ul 10X Titanium Taq buffer
** 13 ul H2O
13 ul H2O
** 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
# 15 sec @ 35 degC
# 15 sec @ 35 degC
# Hold @ 4 degC
# Hold @ 4 degC
----------------------------------
 


* RT rxn:
* RT rxn:
** 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 88: Line 92:
# Hold at 4 degC
# Hold at 4 degC
# Prepare Exo I mix
# Prepare Exo I mix
-----------------------------------


* Exo I mix:
* Exo I mix:
** 2 ul 10X Titanium Taq buffer
2 ul 10X Titanium Taq buffer
** 1 ul 10X Exo III buffer
1 ul 10X Exo III buffer
** 1 ul 0.1 M DTT
1 ul 0.1 M DTT
** 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:
Line 113: Line 117:
# 20 min @ 80 degC
# 20 min @ 80 degC
# Hold @ 4 degC
# Hold @ 4 degC
---------------------------------------------


* PolyA mix:
* PolyA mix:
** 1 ul 10X Titanium Taq buffer
1 ul 10X Titanium Taq buffer
** 1.5 ul 20 mM dATP (dilute 100 mM dATP 1:5)
1.5 ul 20 mM dATP (dilute 100 mM dATP 1:5)
** 1.2 ul 1:5 RNase H
1.2 ul 1:5 RNase H
** 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
# Hold @ 4 degC
# Hold @ 4 degC
------------------------------------


* 2nd strand mix:
* 2nd strand mix:
** 78.25 ul 2x Terra buffer
78.25 ul 2x Terra buffer
** 1 ul 10 uM Tagging primer
1 ul 10 uM Tagging primer
** 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 151: Line 157:


* PCR mix (Clontech):
* PCR mix (Clontech):
** 21.1 ul 2x Terra
21.1 ul 2x Terra
** 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):
** 21.1 ul 2x HiFi U+
21.1 ul 2x HiFi U+
** 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:

Revision as of 21:20, 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