ENCODE and modENCODE
Guidelines For Experiments Generating Data using RNA-Binding Proteins (RBPs):
ENCODE and modENCODE Standards for RIP-Chip and RIP-Seq Experiments
Version 2.0, 9 January 2012

Introduction: In eukaryotic organisms, gene regulatory networks require an additional level of coordinationthat links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed
as passive molecules in the pathway from transcription to translation. However, it is now clear that RNAbinding proteins (RBPs) play a major role in regulating multiple mRNAs in order to facilitate gene expressionpatterns. Several ENCODE/modENCODE researchers are mapping RBP-binding sites using RBP
immunoprecipitation (RIP) followed by extraction of associated RNA and characterization using genomic
microarrays (RIP-Chip), ENCODE Tiled-array (RIP-T-Chip) or by high throughput sequencing (RIP-Seq).
In an effort to facilitate and unify high-quality RBP-based ENCODE/modENCODE data sets, this document aims
to provide uniform standards and guidelines for experiments that map RBP-associated RNA networks and thelocation of RBP-associated binding sites and regulatory elements. These are proposed standards by which tomonitor the quality, reproducibility, specificity, sensitivity and reporting of RIP experiments. We realize that
there is no single path to validating and verifying an experiment, and that these standards may require futurerevision, as technologies change and our understanding of the data increases. We also recognize that there isan inherent trade-off between reporting a complete list of RNA-based functional elements, and a highconfidence list of RNA-based functional elements.
I. General Required Measurements and Procedures• Growth and maintenance of cells. The protocol for growing and maintaining cells should follow thegeneral outline described in the Guidelines For Experiments Generating Chromatin and Transcription
Factor Data, Version 1.0. Briefly, for each experiment, the growth time of cell lines should bedetermined by recording the date and time at which cells were put into culture, and when they wereharvested. Investigators should go back to the original stock after growing a culture for one month.
Passage number should be assessed and recorded for primary cells. Primary cells should not exceed 4passages. Any experiment that does not follow the officially approved protocol for that cell line/typeshould be noted (see metadata standards).
II. Antibody Characterization• Presently, there are a limited number of well-characterized antibodies against RBPs. To ensurespecificity of the antibodies used, the following tests are suggested. The data generated to characterize
the antibody should be made publically available alongside the ENCODE dataset and must be
registered with the DCC prior to submission of the data.
•It is expected that as the number of antibodies increase in the near future, the use of multiple
antibodies against the same protein will become the standard for the field.

•Multiple antibodies, when available, should be used for RIP experiments, and a statistically significant
overlap of targets will be used for characterization. Any reasonable method of correlation can be used(such as r2 greater or equal to 0.5 or 80% of the top 40% of the targets of one list should overlap that of
the list from the second antibody).

RIP-chip, RIP-seq Guidelines V2.0January 20121•For antibodies directed against members of a multi-gene family, antibodies should be directed toprotein regions that are unique to individual family members. Any potential cross-reaction should be
noted when reporting data collected using that antibody.

•Different Lots of antibody from the same manufacturer do not need to be characterized if they
recapitulate previous RIP results to the standards established for biological replicates.

•RIP using epitope-tagged proteins - As there are few antibodies available to RNA binding proteins, RIP
experiments may also be conducted in cell lines expressing epitope-tagged RNA binding proteins. In
these cases, the antibody that recognizes the epitope-tag should be characterized in the same manner
as antibodies that directly recognize RNA binding proteins.
Western blotting and/or
immunofluorescence should be used to monitor the expression level of the epitope-tagged RNA
binding protein. Additionally, Western blotting should monitor the efficiency and specificity of the RIP.

•Immunoblot Analysis. Successful antibodies should show essentially a single band of the predicted sizeby immunoblot analysis of RIP material. The minimum standard to be met is that the predicted
reactive band composes at least half of the total signal in the lane, as assessed by quantitative
immunoblot analysis of RIP-material from whole cells lysate (e.g. quantitative imaging ofchemiluminescence or chemifluorescence).

•Immunofluorescence (IF) Analysis. If immunoblots are unsuccessful, immunofluorescence may be used
as a characterization measure. The immunofluorescence pattern must conform to expectations (forexample, nuclear staining for a chromatin protein or TF).

•To be considered “RIP” quality, an antibody RIP-IP Western blot activity should be reactive against
preferably one prominent band of the appropriate molecular weight. Although multiple bands due toisoform, phosphorylation or glycosilation differences are to be expected. With the exception of theimmunoglobulin heavy and light chain, no other activity should be present and the negative control Ab
or beads alone IP should be negative. If other bands are present, the appropriate target band should
be at least 2-times the intensity of any other band.

•As discussed in the March mod/ENCODE Consortia meeting, documents describing characterization ofantibodies to RBPs, and epitope-tagged RBPs will be required for complete data submission. Effective
March 1, 2011, 1) New antibodies submitted for registration to the DCC must include a completeantibody characterization PDF document (see below template) and 2) New data submissions thatinvolve antibodies must include the registered antibody information or they will not be accepted. For
more information regarding the antibody characterization policy, including a copy of thecharacterization form and a proposed workflow for registering future antibodies, please visit theENCODE Antibodies wiki page: http://genomewiki.ucsc.edu/EncodeDCC/index.php/Antibodies

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RIP-chip and RIP-Seq Data Production• As outlined in the mod/ENCODE Data Verification Standards, RIP-Chip, RIP-T-Chip and RIP-Seq data
should be verified to assess the reproducibility of the experiment by performing at least 2 biologicalreplicates. Three biological replicates are recommended for RIP-Chip and RIP-T-Chip and two for RIPSeq. The replicas should have a significant correlation coefficient when analyzing the top targets on thelist. 80% of the top 40% of the targets of one list should overlap that of the second list. Once data
have been verified, they should be released into public databases as consistent with theENCODE/modENCODE Data Release Policy.
RIP Validation by use of an alternate detection platform• Similar observation using alternative detection methods increases confidence that detected events arereproducible. However alternate methods of detection do not test the biological validity of the results
and may not assess the accuracy of the underlying biological assay. Small-scale validation can beperformed by subjecting individual transcripts of interest to Northern blot following RIP, todemonstrate specificity with respect to other control transcripts not associated with the factor ofinterest. Northern blotting is also advantageous with respect to sequencing and microarray
technologies, as it reports directly on the size(s) of the associated transcripts.
Specificity and Sensitivity• Specificity and sensitivity may be difficult to accurately estimate for RIP experiments since the numberof true positives and negatives is frequently unknown and methods for identification of in vivo binding
independent of RIP are difficult to ascertain. Nonetheless, the positive predictive value (defined asTP/(TP+FP) where TP is true positives and FP is false positive) for RIP experiments may be estimatedusing data comparing RIP-Chip or RIP-Seq results to data available from other sources (e.g. dataobtained using other methods and knowledge from the literature) with the realization that resultsfrom the literature are not necessarily accurate. In addition, depending upon the results, comparisons
to expectations can be made (e.g. binding to known RNA-binding sites and/or motifs) and tested toattempt to ascertain a (potentially biased) false positive rate.

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