Arichard:Notebook/2013/March

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March 2013[edit]

March 1, 2013[edit]

  • Performing RT first strand and second strand synthesis on total RNA samples 1-16 and Vipul's samples 17-25:
Vipul's samples '
1 A3-1, 1 pg
2 A3-2, 1 pg
3 A100, 1 pg
4 A3-1, 10 pg
5 A3-2, 10 pg
6 A100, 10 pg
7 A3-1, 50 pg
8 A3-2, 50 pg
9 A100, 50 pg
  • I will also have 16 samples of total RNA as follows:
Sample RNA (pg) Sample RNA (pg)
1 100 9 10
2 100 10 10
3 10 11 10
4 10 12 10
5 10 13 10
6 10 14 10
7 10 15 NTC
8 10 16 NTC

March 2[edit]

  • Came in to start IVT on samples prepared yesterday. Started at 11 pm, end at 12 noon on March 3

March 3[edit]

  • Mg2+ fragmentation on IVT. 4 ul of NEB Frag Buffer to each 16 ul IVT product. 3 min @ 94 degC, then immediately put on ice and add 0.5 M EDTA.

March 4[edit]

  • Cannot run bioanalyzer yet because the Palsson lab does not have enough samples to fill a chip (11 lanes). I will run IVT products from 2/7 and 2/14 on PAGE to see if they are even visable.

March 5[edit]

  • Lane setup for PAGE on 2/7 and 2/14 IVTs:

(0.5 ul low mass ladder) (2 ul of 2/7 IVT) (15 ul of 2/7 IVT) (2 ul of 2/14 IVT) (15 ul of 2/14 IVT)

File:Ivt mg frag 2 7 and 2 14.jpg

Both the 2 ul and 15 ul lanes were visible for both the high mass (2/7) and low mass (2/14) input IVT reactions. I think I can get away without using the bioanalyzer, which is really nice because the Palsson lab does not run them on a regular enough basis.

I think that I need a purification step before IVT. The T7 primers are single stranded, so they shouldn't be a template for IVT, but the size correlates with what I am seeing on these gels. I will move the T7 primer reaction to before the column cleanup, giving me primer free template for IVT.

Below is the gel for the IVT I ran over the weekend, on both my total RNA and Vipul's purified mRNA

File:Ivt mg frag 03 05 2013.jpg

  • I need to switch to TBU gels when looking at RNA.

March 06[edit]

  • After talking with Rui, I am going to move the T7 primer reaction back before purification, and I am going to switch from EtOH precipitation to the cDNA columns provided with the Ambion kit.
  • It seems like the bright bands at the bottom of my gels are the same size as the common sequence and 5 prime adaptor on the T7 primers, even though the T7 primers should not be acting as a template (single stranded and also the sense strand). Rui said that she notices the same thing when she attempts IVT without removing T7 primers.

March 07[edit]

  • Started new run. Will start IVT tonight, mg frag tomorrow morning followed by PAGE gel.
  • Same setup as before, except for two additional samples (positive and negative controls).
  • Positive control is half volume relative to the Ambion manual, and 5X volume relative to CEL-Seq. Both have 0.5 ul of the T7 primer that was shipped with the kit (unknown concentration). The negative control has 5 ul of UV treated H2O, while the positive control has 5 ul of 100 ng/ul total RNA (from Rui). These two samples will not undergo the Bst reaction to add the T7 primers, since they already have T7 promoter sequences.

March 08[edit]

  • The positive and negative controls look great, with visible difference between fragmented and non fragmented RNA in the positive. The experimental samples were identical to negative control, however.

File:Ivt mg frag 3 08 2013.jpg

March 09[edit]

  • Great meeting with Ben Spike today, from 2 to 4pm. After discussing it, I think that the last IVT failed because I had the wrong buffer conditions for Bst. The second strand reaction via DNA Pol I must use a buffer very close to NEB buffer 2, because DNA Pol I works best in those conditions. Bst also works best in NEB 2. When I added NEB 2 to prepare for the Bst reactions, therefore, I was making the buffer concentration effectively 2X. I'm still not sure that Bst would have zero activity under these conditions, however. Whatever happened lead to literally zero T7 activity on the template, and therefore in order for Bst to be the culprit, it would have to be completely inactive.
  • The previous gels, from 3/05 and 2/07 + 2/14, are probably positive data. There is a visible smear in the right size range. The large band at the bottom is approximately the same size as the T7 primer, which ???????????????? (not sure what to write here, will come back to it in a minute).


Due to the overwhelming abundance of primer relative to sample, this reaction dominates and therefore contributes the majority (approx. 95%) of the template for IVT.

  • There are two approaches to fix this:
    • First, I could do a column cleanup before the Bst reaction instead of an EtOH precipitation. This should remove the primers.
    • Second, I could

March 11[edit]

  • Vipul has ordered a new primer for the CEL-Seq project, identical to barcode #39 from Hashimshony et al. This primer has the 5' adapter and T7 promoter to begin with.
    • It is actually 3 primers that I will use together in order to get the 3' terminal V (A, C, G) on the poly dT stretch.
  • I will use this primer along with our own (using Bst to incorporate the T7 promoter).
  • Vipul needs approximately 15 million reads per library, out of a 40 million theoretical maximum per lane on the GAIIx. I need to make sure that Vipul is aware that CEL-seq requires paired end sequencing, which means that he may have to wait a while to get in on a run.
  • Vipul also want to do an Ampure XP bead purification on cDNA before IVT, rather than the cDNA column included with the kit. This does not make sense to me, since he is comparing his own method to CEL-Seq, and therefore improving or otherwise modifying the CEL-Seq protocol would not give any advantage in answering his reviewers comments.

March 13[edit]

  • Vipul gave me the new primers in 3 tubes (3' terminal A, C, and G). Each primer is 100 pmol/ul, to be mixed 1:1:1.
  • I also have new samples of mRNA: A3(1:100) at 500 pg/ul and A100(1:100) at 1000 pg/ul. He wants both 50 and 100 pg libraries made from each, with technical replicates for A100, 6 samples total.
    • A3 - 50 picogram
    • A3 - 100 picogram
    • A100 - 50 picogram , Technical Replicate 1
    • A100 - 50 picogram , Technical Replicate 2
    • A100 - 100 picogram , Technical Replicate 1
    • A100 - 100 picogram , Technical Replicate 2

March 14[edit]

  • Because I have only one barcode to work with on Vipul's samples (#39 from CEL-Seq paper), I will have to do all 6 samples separately.
  • I am going to process Vipul's samples tomorrow, March 15. I am confident that the long primer will work, because the control primer from the kit (Poly dT - T7 promoter) worked well on the last run. This will give me a chance to move forward with library creation using the TruSeq kit. The Bst reaction will have to wait until next week, or this weekend.
  • How much primer and ERCC should I use?
    • I can mix mRNA, primer, and ERCC together for A3 and A100 --> 2 master mixes for 100 pg, which I can then dilute for 50 pg.
    • mRNA should represent only 5% of total RNA from the cell.
    • 100 pg mRNA --> 2000 pg (2 ng) total RNA
    • CEL-Seq uses .165 pmol of primer per 10 pg total RNA --> 32.9 pmol per 2 ng total RNA (equivalent to 100 pg mRNA).
  • I will dilute 1000 pg/ul A100 1:1 with H2O to make 500 pg/ul, such that the input volume will be the same as A3 (which is 500 pg/ul).
  • I need 100 pg of mRNA in 1.2 ul, times 2 for replicate, plus another 2 ul to dilute to 50 pg level = 1.2 x 2 + 2 = 4.4 ul --> Make 5 ul, containing 417 pg mRNA, which corresponds to 8.34 ng total RNA.
    • 417 pg mRNA --> 0.834 ul of 500 pg/ul A3 and A100.
    • In this 5 ul, I need 138 pmol of primer --> 1.38 ul of 100 pmol/ul primer mix.
    • In this 5 ul, I also need 1.67 ul of 1:10000 ERCC.
  • Sample setup mix for both A3 and A100:
    • 0.834 ul 500 pg/ul mRNA.
    • 1.38 ul 100 pmol/ul primer.
    • 1.67 ul 1:10000 ERCC.
    • 1.12 ul UV-treated H2O.
  • Dilute 2 ul of this mix with 2 ul H2O to make 4 ul of 50 pg/ul mRNA.
  • Aliquot 1.2 ul of each mix into the following 6 0.2 ml low retention tubes:
    • A3_50pg.
    • A3_100pg.
    • A100_50pg_1.
    • A100_50pg__2.
    • A100_100pg_1.
    • A100_100pg_2.
  • From this point on, the protocol is the same as CEL-Seq.

  • I just met with Vipul again (3:20-4:00p), and he wants to change the experimental setup.
    • First, he wants to use another barcode (#40 from CEL-Seq).
    • Second, he wants a 10 pg condition for both A3 and A100, with A100 in duplicate as always. This gives a total of 9 samples.
    • Third, he does not need ERCC spike in controls for his samples, as he has already sequenced them using other methods and therefore will know a priori both the relative and absolute transcript abundances (RPKM and TPM values for all transcripts).
  • We have agreed on the following sample setup:


Sample Input (pg) Barcode # Lane/IVT rxn
A3 100 39 1
A3 50 39 2
A3 10 39 3
A100 100 40 1
A100 50 40 2
A100 10 40 3
A100 100 39 4
A100 50 39 5
A100 10 40 5


  • No less than 5 ul pipeting volume to ensure accuracy.
  • My lowest IVT input is IVT rxn #5, which has 10 + 50 = 60 pg mRNA input. This is equivalent to 60/0.05 = 1200 pg total RNA, which is above the 400 pg lower input threshold for IVT.
  • I will have 3 input levels: 100 pg, 50 pg, and 10 pg mRNA:
    • 100 pg mRNA in 1.2 ul --> 83.3 pg/ul mRNA.
    • 50 pg mRNA in 1.2 ul --> 41.7 pg/ul mRNA
    • 10 pg mRNA in 1.2 ul --> 8.33 pg/ul mRNA.
  • I need 16.5 pmol of primer per 1 ng of total RNA, and mRNA = 0.05*total RNA --> 330 pmol primer per 1 ng mRNA. Scaling linearly, this gives the following:
    • 33 pmol primer per 100 pg mRNA in 1.2 ul --> 27.5 pmol/ul primer.
    • 16.5 pmol primer per 50 pg mRNA in 1.2 ul --> 13.8 pmol/ul primer.
    • 3.3 pmol primer per 10 pg mRNA in 1.2 ul --> 2.75 pmol/ul primer.
  • I will dilute both mRNA and primer to 2X concentration, then mix 5 ul of mRNA with 5 ul of corresponding primer to give 10 ul of mix, of which I will use 1.2 ul per reaction.
    • 2 X 83.3 pg/ul = 167 pg/ul mRNA.
    • 2 X 41.7 pg/ul = 83.3 pg/ul mRNA.
    • 2 X 8.33 pg/ul = 16.7 pg/ul mRNA.
    • 2 X 27.5 pmol/ul = 55 pmol/ul primer.
    • 2 X 13.8 pmol/ul = 27.5 pmol/ul primer.
    • 2 X 2.75 pmol/ul = 5.5 pmol/ul primer.
  1. Dilute 500 pg/ul A3 to 167 pg/ul:
    1. Add 20 ul to 40 ul H2O --> 60 ul 167 pg/ul A3.
  2. Dilute 167 pg/ul A3 to 83.3 pg/ul:
    1. Add 20 ul to 20 ul H2O --> 40 ul 83.3 pg/ul A3.
  3. Dilute 83.3 pg/ul A3 to 16.7 pg/ul:
    1. Add 20 ul to 80 ul H2O --> 100 ul 16.7 pg/ul A3.
  4. Dilute 1000 pg/ul A100 to 167 pg/ul:
    1. Add 20 ul to 100 ul H2O --> 120 ul 167 pg/ul A100.
  5. Dilute 167 pg/ul A100 to 83.3 pg/ul:
    1. Add 20 ul to 10 ul H2O --> 40 ul 83.3 pg/ul A100.
  6. Dilute 83.3 pg/ul A100 to 16.7 pg/ul:
    1. Add 20 ul to 80 ul H2O --> 100 ul 16.7 pg/ul A100.
  7. Mix the three primers for barcode #39 (3' terminal A, C, and G), 100:100:100 ul.
  8. Do the same for primer barcode #40.
  9. Dilute 100 pmol/ul primers to 55 pmol/ul:
    1. Add 20 ul to 16.36 ul H2O --> 36.4 ul 55 pmol/ul primer.
  10. Dilute 55 pmol/ul primers to 27.5 pmol/ul:
    1. Add 20 ul to 20 ul H2O --> 40 ul 27.5 pmol/ul primer.
  11. Dilute 27.5 pmol/ul primers to 5.5 pmol/ul:
    1. Add 20 ul to 80 ul H2O --> 100 ul 5.5 pmol/ul primer.
  12. Mix the following, 5 ul + 5 ul each:
Sample # pg/ul A3 (5 ul) pg/ul A100 (5 ul) pmol/ul primer #39 (5 ul) pmol/ul primer #40 (5 ul)
1 167 N/A 55 N/A
2 83.3 N/A 27.5 N/A
3 16.7 N/A 5.5 N/A
4 N/A 167 N/A 55
5 N/A 83.3 N/A 27.5
6 N/A 16.7 N/A 5.5
7 N/A 167 55 N/A
8 N/A 83.3 27.5 N/A
9 N/A 16.7 N/A 5.5
  • I could/should still do 1 sample (either A3 or A100) using my primers, with a bead purification before Bst rxn.

March 19[edit]

File:2013 03 19 ivt mg frag.jpg