[Maflores11]

Hi Kathi_seq,

This is Miguel Flores (user: ashes11), I was unable to login in with my previous account.

In order to minimize COCl2 precipitation (likely due to DTT in NEB4 Buffer), I tried less concentration of COCl2 until I found a balance. I recommend to run an experiment with titrations of COCl2. I should probably contact my former adviser to check my notes to get specifics. LinDA worked in my hands, but requires some tweaking.

Cheers!

[Kathi_seq]

Thanks Daytwa for your swift reply.

[Daytwa]

Sorry! It never worked for us.

[Kathi_seq]

Anyone actively using LinDA?

Hey,

I am struggling with the LinDA-protocol. Ashes11, did you find an alternative for the NEB4 buffer?
I have seen the brown precipitate after in vitro transcription.

I know these posts are pretty old, but maybe I am lucky and someone is still active here?

Thanks for your help in advance!

[ashes11]

It seems that the DTT in NEB Buffer 4 precipitates COCl2 in the reaction solution. T-tailing of an internal control (50 ng PCR fragment, 360 bp) does not work in my hands when using NEB Buffer 4, 5mM COCl2 and NEB TdT enzyme. Here more proof from Schreiber Lab:

http://www.broadinstitute.org/chembio/lab_schreiber/pubs/protocols/IVT_Supplement/FAQ.html

[Daytwa]

Has anyone made any progress with this protocol? We can't yield higher than ~10 ng of RNA.

[MolBioAsk]

Hi,

I am struggling with LinDA now, and thanks for all your helpful suggestions and ideas on the subject, especially from Linampli.

I used my ChIP pulled down chromatin 2ng(maybe nanodrop not acurate) to do LinDA and got specific amplification, total amount 180ng; and when I use 5opg of pulled down DNA for LinDA, I got low yield non-specific amplification.I think I should start with accurate 50pg of Input DNA to set up the experiment first.

Here are my questions:
1. 50pg of sonicated chromatin(100bp to 1kb) and 50pg of 250bp DNA fragment is different. Did someone get ideal amplification from 50pg of sonicated chromatin?

2. Several post above said they got lower efficiency of T-tailing and oligo T7-annealing. how to improve the efficiency, other than usage of TSAP, eg, increase reaction time, increase the enzyme de-activation temparature..or..?

Hope to get some help here. Thanks a lot.

[linampli]

hi,
to get back to the library prep, I am now finalising a procedure utilising LinDA for Illumina library prep. With the modified protocol one should get an Illumina library directly with LinDA, without using PCR cycles as is the case with NuGene. Basically I incorporate the Illumina primers in the RT and 2nd strand synthesis steps.

Hi, so I think I'm managing to get it to work. After amplifying 50pg I got back 48ng which is more than I expected. Unfortunately when I try to use this DNA for qPCR validation some primer sets give my much higher ct values than others. It looks as if there is some kind of a bias introduced but it doesn't seem to be dependent on GC. Any idea what this might be?
I'm also thinking about the kit I want to use to construct the libraries. Illumina's ChIP-seq seems to be laborious. Any thoughts about the new kits like NextFLEX ChIP seq or the NuGene low input library preparation kit (ready for 1ng of DNA)?

[linampli]

hi,
Did you purify the DNA after LinDA with Qiagen columns or SPRI beads? I found that the T7 poly A primer doesnt get completely removed with column purification, this leads to interference in qPCR. SPRI beads purification (eg. Ampure) manages to remove the primers efficiently and qPCRs improve significantly.

Hi, so I think I'm managing to get it to work. After amplifying 50pg I got back 48ng which is more than I expected. Unfortunately when I try to use this DNA for qPCR validation some primer sets give my much higher ct values than others. It looks as if there is some kind of a bias introduced but it doesn't seem to be dependent on GC. Any idea what this might be?
I'm also thinking about the kit I want to use to construct the libraries. Illumina's ChIP-seq seems to be laborious. Any thoughts about the new kits like NextFLEX ChIP seq or the NuGene low input library preparation kit (ready for 1ng of DNA)?

[a3911145]

LinDA

Hi, I have seem the same phenomenon in the qPCR. But whether it's a bias or not, is under question. If the amount of gene B is higher in the starting material, it may amplify more after LinDA than A. I think you can try to compare the amplification ratio of one gene between specific antibody group and IgG control group.
for instance, gene A in specific antibody group is originally "10", then after LinDA is "100"; gene A in IgG control group is "1", then after LinDA is "9", the ratio of amplification ratio is 10/9. gene B in specific antibody group is originally "20", then after LinDA is "400"; gene B in IgG control group is "2", then after LinDA is "38", the amplification ratio is 20/19. If that so, I think it may not influence result of the ChIP-seq.
I'm glad to hear that another people have got the protocol to work. However, I am still have trouble with ‘T’ tailing and attaching T7 primer. I follows the protocol except that using another ThermoSensitive Alkaline Phosphatase(Fermentas) and inactivating at 75° 5 min according to the user manual. Can you share some experience of your LinDA?
thanks in advance！

Hi, so I think I'm managing to get it to work. After amplifying 50pg I got back 48ng which is more than I expected. Unfortunately when I try to use this DNA for qPCR validation some primer sets give my much higher ct values than others. It looks as if there is some kind of a bias introduced but it doesn't seem to be dependent on GC. Any idea what this might be?
I'm also thinking about the kit I want to use to construct the libraries. Illumina's ChIP-seq seems to be laborious. Any thoughts about the new kits like NextFLEX ChIP seq or the NuGene low input library preparation kit (ready for 1ng of DNA)?

[epignome]

LinDA

Hi, so I think I'm managing to get it to work. After amplifying 50pg I got back 48ng which is more than I expected. Unfortunately when I try to use this DNA for qPCR validation some primer sets give my much higher ct values than others. It looks as if there is some kind of a bias introduced but it doesn't seem to be dependent on GC. Any idea what this might be?
I'm also thinking about the kit I want to use to construct the libraries. Illumina's ChIP-seq seems to be laborious. Any thoughts about the new kits like NextFLEX ChIP seq or the NuGene low input library preparation kit (ready for 1ng of DNA)?

[a3911145]

I am a PHD student from China
Similar to thedavid, I think I have the same problem in LinDA: very low efficiency of the‘T’ tailing or low efficiency of attaching T7 primer.
I choose another Shrimp alkaline phosphatase produced by Fermentas. I have succeeded in LinDA for only one time, when I add dTTP without ddCTP, and inactivated Klenow at 75° for 20min (start with 50ng DNA [input DNA, fragmented by Ultrasound] produces 17000ng RNA and finally 5000ng DNA). But when repeat the experiment, I failed again and again, with no RNA yield from 20-50ng DNA starting material and no ampification of ChIP-DNA confirmed by realtime PCR.
Working hard on it for over two months, I'm now can confirm that this protocol can work, but I don't understand why it is so hard to stabilize the system!
Has anyone not in Hinrich Gronemeyer's lab gotten this process to work?
Has anyone know why the efficiency of ‘T’ tailing/attaching T7 primer is low?
Thanks a lot!

[linampli]

someone pointed out a mistake in the amplification protocol, in the reagent list it is given as 1mM of rATP, rGTP, rCTP, rUTP. It should be 100 mM each as is supplied by the kit, please do not adjust the concentration.

[thedavid]

1. Thanks, I'll give it a try.
2. Sorry, I wasn't clear - if the Klenow was added, then 10 mM dNTPs were added. What I meant was that 100 uM dNTPs were added instead of 100 uM T-mix. As a control, of course, since they wouldn't be very useful for adding the T7-BpmI-OligoA(15)
3. Perhaps.
4. That's true - I was using 50ng because it is difficult to visualize less on a gel.
5. We don't have a Qubit, but we do have BioAnalyzer Pico chips, which can measure in the hundreds of picograms of RNA.
6. Yes, I was using the SS III kit with the 10x buffer (hence I could use 26ul of RNA instead of 22ul), since that is what I had on hand. I'll switch to the kit with the 5x buffer in the future.

If you only expect 20ng of RNA total from 50pg of DNA, then I should expect to get only around 5-10ng of DNA in the end? Which would not be a challenging amount to build a library for next-gen sequencing. So I would need at around 200 pg of input into the system to comfortably generate enough material for next-gen sequencing?

the detailed protocol is in nature protocols where the exact reaction volumes are given.
1. Unfortunately Promega seems to have discontinued SAP, however, the thermostable phosphatase seems to be a good alternative.
2. In your gel you mention that NO dNTP was added for the reactions in
lanes 1 2, 3. Is that right? If there is no dNTP added with the T7 oligo klenow would start chewing back the strand.
3. Perhaps there is something wrong with your SAP. The SAP step is expendable, ie. it is there only to improve the efficiency.
4. Try your reaction with 1 ng of DNA, 50 ng maybe too much substrate and that is why you see only 50% efficiency.
5. Do a Qubit HS for RNA quantitation, Qubit seems to be the most accurate and sensitive test for DNA/RNA. For 50 pg of substrate you should get around 10 to 20 ng of RNA (in 30 µl), ie. 0.3 to 0.6 ng / µl (nanodrop volume). this is far below the detection limit for nanodrop or for that matter bioanalyser.
6. so try Qubit, and elute in 20 µl (imp for the next step)

[linampli]

the detailed protocol is in nature protocols where the exact reaction volumes are given.
1. Unfortunately Promega seems to have discontinued SAP, however, the thermostable phosphatase seems to be a good alternative.
2. In your gel you mention that NO dNTP was added for the reactions in
lanes 1 2, 3. Is that right? If there is no dNTP added with the T7 oligo klenow would start chewing back the strand.
3. Perhaps there is something wrong with your SAP. The SAP step is expendable, ie. it is there only to improve the efficiency.
4. Try your reaction with 1 ng of DNA, 50 ng maybe too much substrate and that is why you see only 50% efficiency.
5. Do a Qubit HS for RNA quantitation, Qubit seems to be the most accurate and sensitive test for DNA/RNA. For 50 pg of substrate you should get around 10 to 20 ng of RNA (in 30 µl), ie. 0.3 to 0.6 ng / µl (nanodrop volume). this is far below the detection limit for nanodrop or for that matter bioanalyser.
6. so try Qubit, and elute in 20 µl (imp for the next step)