Arichard:Notebook/2013/May

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May 2013[edit]

May 7, 2013[edit]

  • Began CEL-Seq with 1 ng UHRR, 16 pmol primer, 0.4 ul 1:1e4 ERCC in a single reaction, to be split before IVT to give a negative control.
100 uM primer #38 1 ul
1:1e4 ERCC 2 ul
5 ng/ul UHRR 1 ul
2 ul H2O 2 ul
Total 6 ul
  • Used 1.2 ul of this mix. RT and 2nd strand as normal.
  • Used Agencourt RNAClean XP beads (1:1 vol:vol --> 10 ul) after 2nd strand. Eluted in 12.8 ul H2O but did not remove from beads. Took 6.4 for negative control.
  • Ran IVT on beads. Negative control received 9.6 ul H2O instead of IVT mix.

May 8, 2013[edit]

File:2013 05 08 ivt test on rna clean beads.jpg

May 9, 2013[edit]

  • pPCR to validate primers: T7 for, hRplp0 rev, ERCC 83 rev, ERCC 108 rev, and ERCC 130 rev. See gel image for rxn details.

May 10, 2013[edit]

File:2013 05 10 primer validation hrplp0 ercc83 ercc108 ercc130.jpg

  • Jeff suggested that I raise the extension temperature to 72 degC, since the KAPA SYBR Fast polymerase is Taq-based. My anneal/extend temp at 60 degC gives the primers too much time to anneal non-specifically.

May 13, 2013[edit]

  • 2nd attempt to validate qPCR primers with hotter extension.
  • No NTCs this time, because they were totally clean last time.
  • I only have enough of the negative control (no polymerase) cDNA left for one more qPCR experiment, about 2 ul containing approximately 1-2 pg.
  • I could still use the positive control cDNA, although it will contain RNA from IVT which may act as forward primer.
  • See gel image for rxn details.

May 14, 2013[edit]

File:2013 05 14 primer validation 2nd try.jpg

  • This gel was overloaded. I will run it again with only 1 ul this time (versus 8.5 ul).

File:2013 05 14 primer validation 2nd try lower loading.jpg

  • Ben Spike is curious whether I have been successful with adapter ligation. I haven't sequenced any libraries, so I can't say for certain. The PCR at the end of CEL-Seq depends on the presence of the adapter, and I have gotten that to work about 50-60% of the time. All of the failures occurred in a two week period while I was working with Vipul.
  • I need a way to confirm for Ben that the libraries I have made have the correct adapters, but PCR would just use the same primers that I used to make the libraries (priming against the 5' and 3' adapter sequences), so it wouldn't really tell me anything new.
  • The only way to confirm that I have usable libraries will be to sequence one. Right now I only have a UHRR library prepared, without spike in. I haven't sequenced it because it was only a validation run on control RNA, but now I think it would be very useful to know that the adapter sequences were incorporated correctly.

May 16, 2013[edit]

  • Submitted UHRR CEL-Seq library to Alan for PE sequencing. Will be run probably 2-3 weeks from now.
  • Leaving for vacation. Return May 29.

May 29, 2013[edit]

  • Single cell isolations on lymphocytes:
    • Spent 2.5 hours this morning to get 2 cells, the second of which is questionable.
    • First hour was spent getting the right cell dilutions, ETDA concentration, and camera settings.

File:2013 05 29 lymphocyte1.tif Lymphocyte #1

File:2013 05 29 lymphocyte2.tif Lymphocyte #2

    • Will continue tomorrow. I need 7 or 8 single cells, plus one pool of ~10 cells.
    • Later this week I will make libraries for these samples.
  • Today after lab meeting I will continue working on a transcript matrix for the single cell SMART-Seq data. So far I have about 8 datasets, but I need a faster way of collecting the information.
  • Once I have the data extracted from the files on genome miner, I will do PCA.

May 30, 2013[edit]

  • I isolated 2 more cells this morning, using smaller pipettes, still using pull = 600 on the puller.
    • Yesterday I used heat = 700. Today I used heat = 800.
  • I also made more careful dilutions today, since I already know the ballpark concentration from yesterday.
    • Diluted 200 ul cells in tube with 800 ul PBS (1:5), then diluted 100 ul of this with 1000 ul of PBS plus 100 ul 0.5 M EDTA (1:12) --> 1:60 dilution total, with 0.042 M EDTA.
    • Each 3 ul droplet of this solution contained ~30 cells --> 1e4 cells/ml is a good working concentration.

File:2013 05 29 lymphocyte3.tif Lymphocyte #3

File:2013 05 29 lymphocyte4before.tif Image of the glass top tube before expelling lymphocyte #4.

File:2013 05 29 lymphocyte4.tif Lymphocyte #4

  • The quality of these images is obviously questionable. It is very difficult to determine the presence or absence of a single intact cell without any kind of stain or dye. One option is to use trypan blue, which should be excluded from live cells and therefore won't interfere with RT primer annealing.
    • Jeff suspects that DAPI interferes with MDA, which is one of the reasons I have been avoiding any kind of staining.
  • At this point, I am strongly leaning towards FACS to sort single cells into a 96 well plate.
    • If I could sort into RT primer annealing solution, then I wouldn't need to worry about excess volume.
    • Blue told me that he has successfully sorted into 2 ul, so 1.2 ul isn't too far off the mark.
    • 96 well is probably preferable to 384, because it is more likely that the cell with hit the bottom of the plate rather than stick to the side somewhere.
  • I need to design a primer to check the library quality before sequencing. I can still used the same gene specific primers I designed for the pre-IVT check, but I need a new forward primer.
    • I can used the 3' adapter region.