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  • #46
    I'm using the TruSeq kit with ChIP-Seq routinely with good success, if anyone wants to give it a go using the kit instead of homemade components. I just use a 1:10 dilution of adapter for ~ 100 ng of sonicated WCE (or 1:50 for IPs) and do the PCR *prior to* gel size selection. I have a Pippin Prep for size selection.

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    • #47
      Kalyan,
      That is an interesting trick to shorten the protocol. I worry a little bit about the efficiency as Klenow is not that great at blunting ends especially at elevated temperatures. For making plasmids it is always suggested that the incubation be done at room temperature. It clearly works but I wonder what the efficiency is compared to a two step protocol.

      Illumina A-tails and then proceeds to ligation without a clean up step. I'd like to give that a try but the protocol is working well so I haven't much of a drive to try it or figure out the conditions. Does has anyone have this working? Perhaps this decreases adapter dimer formation as well.
      --------------
      Ethan

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      • #48
        Hi Ethanol,

        thanks for sharing your experiences and your protocol. It seems very promising. I am wondering if you have repeated any hiseq runs till now and got more than 50 mio reads? Also, how many clusters you got in the first run, and how many of the reads mapped to your reference genome. ... just to get an idea what could be the issue that caused the low number of reads. ( was a bit unclear from your blog)

        Just for general information: meanwhile I had 3-4 attempts using the inline adapter approach from Lefrancois on hiSeq and also GAII. anyways, on the hiSeq I ended up with ~100 mio mapped reads out of ~200 clusters. However, this always required equimolar pools of 4 adapters to avoid possible problems due to biasing. I am still trying to figure out where the loss comes from.
        ... hope that is useful to anyone.

        best, hel

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        • #49
          The low number of reads came from the leftover PCR oligos interfering with the binding of the library to the flow cell. This is fixed in the current version by purifying the final PCR with Ampure XP beads. Of course this is an issue known for years but what could I do, when it took Beckman-Culture months to figure out how fulfill an order to Greece.

          I would still load a little more library then you would with a library made with Illumina's oligos as there seems to be a little magic something with the Illumina oligos. Now if Illumina would be nice enough to send me one of their TruSeq kits, I could figure this out better but again I've been waiting 2 months for a TruSeq RNA kit.

          In my first run the percentages of mapped reads was good (something like 80%), the sequence quality was much better then the other samples on the same run (most likely due to the low cluster density). The cluster formed to successful cluster ratio was very high. These stats have stayed good in subsequent runs using the Ampure XP beads but with more reads.

          My last library didn't go so well. It took 20 cycles total to amplify. Probably just not enough starting material, but annoying. It seems chromatin sheared with micrococcal libraries up more efficiently then chromatin sheared by sonication, but I have no side by side comparison here so it is speculative.
          --------------
          Ethan

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          • #50
            Thanks,
            regarding the leftover oligos - I re-run the libraries on a gel to get rid of them. Works perfect - actually better than with beads since my self annealed oligos are bigger than 100 (for whatever reason), and there is plenty of library material left to run 10 lanes.
            I may have missed that in your protocol, but you are not using 5mC in your oligos right (might blow up the costs x10)?
            Regarding the original illumina oligos, I've heard numerous problems in the ligation step using ChiP material, so people had to use ridiculous amounts of 50ng or above to get a decent library (which requires 4-5 pooled ChIPs) - I don't know if this is due to the methylation, the magic chemistry behind clustering, the size or a combination of all.

            cheers, hel

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            • #51
              Hi everyone-
              Has anyone figured out how little starting material one can use with Ethan's prep and still get enough library? Do you really have to use 10 ng starting material? I have seen protocols claiming to work down to 0.5 ng of starting material.

              Thanks,
              Joe

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              • #52
                We follow the protocol but with 5ng of ChIP DNA (Quantitation by Qubit), we did one at 2.5ng and it also worked. All with a total of 11 cycles of amplification; 4 pre-gel separation, 7 post gel excision) using Kapa HiFi.

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                • #53
                  I've gone with as little as 2 ng and it worked out o.k. I can't remember the exact number of cycles though.
                  --------------
                  Ethan

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                  • #54
                    Hi every one! It is my first post

                    I am very glad to see this topic. As our ChIP samples almost <5ng, the libraries prepared has very low concentration ~1ng/ul (Qubit HS) and always poor quality( high ratio of adapter dimer). Has anyone know the proper criterions for ChIP-seq libary?

                    Other request is that I failed to open the ETHANol protocol links http://ethanomics.files.wordpress.co...ip_truseq3.pdf or http://ethanomics.wordpress.com/. I'll feel so grateful if anyone can send the detail protocol to my email, [email protected] or [email protected]

                    Thanks,
                    lhz

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                    • #55
                      Originally posted by lihzhou View Post
                      Hi every one! It is my first post

                      I am very glad to see this topic. As our ChIP samples almost <5ng, the libraries prepared has very low concentration ~1ng/ul (Qubit HS) and always poor quality( high ratio of adapter dimer). Has anyone know the proper criterions for ChIP-seq libary?

                      Other request is that I failed to open the ETHANol protocol links http://ethanomics.files.wordpress.co...ip_truseq3.pdf or http://ethanomics.wordpress.com/. I'll feel so grateful if anyone can send the detail protocol to my email, [email protected] or [email protected]

                      Thanks,
                      lhz
                      Our version of Ethan's protocol can be downloaded from http://www.keatslab.org/protocols

                      Comment


                      • #56
                        Wordpress.com has some problems. If you just keep clicking reload a bunch of times it will eventually load. Annoying and I'd like to move my blog somewhere more reliable but it never makes it up to the level of importance of something that actually gets done.

                        5 ng is doable. Sub 5 ng is find is usually pushing it.
                        --------------
                        Ethan

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                        • #57
                          Hello, ETHANol and Jon_Keats

                          Thanks very much for replies!

                          I've succeded to download the protocol from http://www.keatslab.org/protocols and will try to prepare my libary again

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                          • #58
                            Minimum molar Requirement for ChIP-Seq libraries

                            Hey ETHANol,

                            This is my first post here and I'd first like to begin that your ChIP-Seq protocol at Ethanomics is religiously followed in my lab. :P

                            I have a slight conundrum however with some of the libraries I have prepared using this protocol of late. Since the ChIP samples I am working with are low in concentration due to the weak antibody, after 'successful' library preparation I am able to obtain a range of 2-10 nM of library sample for my ChIP samples.

                            I wish to know if this would be good enough to allow successful sequencing. The spread of the DNA ranges from 250 - 400 bp which normally denotes a decent library, however I am slightly worried about the concentrations though.

                            We will probably use a HiSeq 4000 for sequencing with 12 samples in a lane. In your expert opinion, do you think this concentration should enable decent sequencing for me?

                            warriert

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                            • #59
                              The BioDynmi ChIP-Seq Library Prep Kit (illumina TruSeq platform) was developed for the construction of high quality libraries using 5 ng to 400 ng of ChIP DNA as input.

                              Three index types are available for the kit:
                              • Non-index (Cat.# 30032S and 30032L): Libraries do not have index.
                              • Single Index (Cat.# 30034S and 30034L): Each of our index primers contains a unique barcode sequence with 6 bases that can be used to identify libraries. Library multiplexing up to 48 samples is possible.
                              • Unique Dual Index (Cat.# 30036S and 30036L): Library multiplexing up to 96 samples is possible with unique dual indexes. We have developed a 4-Base Difference Index System. The system allows us to make indexes that have at least 4 bases different from each other in the 8 bases index length. Our unique dual indexing primers remove sequencing errors such as index hopping, index cross-contamination, mis-assignment of reads, amplification errors, and de-multiplexing errors. The primer set includes 96 pre-mixed unique pairs of i5 and i7 index primers in a 96-well plate.


                              Features
                              • Fast
                                • Total time: 1.5 hrs
                                • Hands-on time: 10 min
                              • Easy procedure
                                • Ready-to-use master mix
                                • Less reaction components
                              • Less magnetic beads required: Reduced more than 50%
                              • Guaranteed quality: Higher library conversion efficiency
                              • Input ChIP DNA: From 5 ng to 400 ng


                              More details: BioDynami ChIP-Seq Library Prep

                              _
                              Last edited by BioDynami; 09-29-2021, 05:45 PM.

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