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Protocols

Word to the wise: A lot of the material on this blog is produced while I am at home, which means there is most likely a baby that wants to be the next Steve Jobs trying to bang on my keyboard and a bored dog biting my feet while I am writing…so if something doesn’t look right in a protocol, it probably isn’t. If you spot a mistake, send me an e-mail at eford.dna-at-gmail-dot-com and I’ll fix it.

<a title="NEW PROTOCOL PAGE ON GOOGLE DRIVE" href="https://drive.google.com/folderview?id=0B4m8jK-CZSGmbTNpMVhndm9RWGs&usp=sharing

Ribo-Zero rRNA removal for RNA-seq with FFPE samples

TruSeq RNA Sample Prep Kit v2 using one-third sample volumes

Native ChIP protocol

NGS qPCR quantitation protocol

NGS PCR cycle quantitation protocol

MeDIP-seq protocol with TruSeq adapters

ChIP-Seq library construction using the Illumina TruSeq adapters

ChIP (MNase fragmentation) protocol

ChIP (fragmentation with Covaris) protocol

ChIP (fragmentation in 0.1% SDS – tip sonicator) protocol

ChIP (fragmentation in 1% SDS – tip sonicator) protocol

Quick ChIP (MNase fragmentation) protocol

ChIP Primer Design

Antigen production protocol

Antibody purification protocol

Immunostaining protocol

4% paraformaldehyde

Lentivirus transduction protocol

Immunoprecipitation of chromatin associated protein complexes

Minipreps

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3 Comments
  1. x.zhouustc@gmail.com permalink

    Hi, Ethan,

    I am wondering if you have tried RRBS-seq with Truseq adapters. From the illumina website:

    TruSeq adapters are methylated. However, the kits are not currently compatible for the following reasons:
    WGBS: The PMM (polymerase mix) used in the TruSeq DNA Sample Prep kits cannot handle uracil bases in its template. This means that bisulfite-converted DNA cannot be processed directly with TruSeq kits, unless another polymerase is used to amplify the samples.
    RRBS: Same as above, and in addition the length of TruSeq adaptors results in the generation of adaptor dimers that overlap the size range of the targeted DNA, making them incompatible with this application.

    What do you think about it? Do you know if anyone has tried the Truseq adapters for RRBS?
    Thanks a lot!
    Josie

  2. Robbie Sierra permalink

    Would you happen to have a protocol for qPCR analysis of ChIP target DNA? There seems to be a couple different methods (% Input or fold change) and Im not entirely sure how to appropriately setup the qPCR for this analysis.

    Thanks.

  3. Hi Ethan,
    I’m wondering if you could comment briefly on the difference between the standard and quick MNase ChIP protocols — do you find the quick method produces similar results? In particular the 6x wash step stands out — i.e. using the LiCl buffer six times instead of the low salt, high salt, and then the LiCl buffer. Also I see some commercial ChIP kits are using faster reverse-crosslinking (e.g. 30 min at 95oC) — have you tried these and chosen to stick with the more traditional 4h to overnight at 65oC? Also any objections to including the proteinase K with the reverse crosslink?
    Thanks for any additional info!
    Best wishes
    CK, London, UK

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