Arichard:Notebook/2013/June

From ZhangLabWiki
Jump to navigation Jump to search

June 2013[edit]

June 2, 2013[edit]

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 25, 2013[edit]

  • 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[edit]

  • 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

Sequences used in Quartz protocol:

Name Sequence
RT primer TATAGAATTCGCGGCCGCTCGCGATAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTTTTT
Tagging primer TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT
Suppression PCR primer GTATAGAATTCGCGGCCGCTCGCGAT


  • 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[edit]

  • 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).

I used NEBNext Index Primers (the loop adapters). The following is directly from the NEBNext Multiplex Oligos for Illumina manual.

Product Index Primer Sequence Expected Index Primer Sequence Read
NEBNext Index 11 Primer for Illumina 5´-CAAGCAGAAGACGGCATACGAGAT[GTAGCC]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´ GGCTAC
NEBNext Index 12 Primer for Illumina 5´-CAAGCAGAAGACGGCATACGAGAT[TACAAG]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´ CTTGTA

I used Index 12 for sample A1 (Terra) and Index 11 for Sample A2 (KAPA HiFi Uracil+).


CORE fragmentation and library prep[edit]

  • 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

I size selected the KAPA library (Index 11) and gave it to Alan for sequencing (AR_QC_500pgUhrrKapa_Jun26).

June 27, 2013[edit]