1. Additional high-throughput sequencing techniques (finding all functional elements of genome)
June 15, 2017

3. DNA binding protein-DNA interactions
CHIP-seq
Chromatin Immunoprecipitation followed by HTS
Used to identify enhancer and TF binding sites as well as histone modification sites
Requires antibody to DNA binding protein
DNAase-seq
identify the location of regulatory regions, based on the genome-wide sequencing of regions sensitive to cleavage by DNase I.
FAIRE-seq
Formaldehyde-Assisted Isolation of Regulatory Elements
formaldehyde cross-linking more efficient in nucleosome-bound DNA than in nucleosome-depleted DNA

4. CHiP-Seq Generally identifies binding sites +/- 300 bp
Contaminating DNA is an issue

5. ChIP-exo Variant of nuclease protection assay
Exonuclease digests only DNA that is not bound
More specificity and high detection of weakly bound proteins
Can use eptiope-tagged proteins for the immunoprecipitation, but a good antibody remains a limitation

6. DNAase-seq
Identify regulatory or transcriptinally active regions based on accessibility to DNase I
Requires permeabilization of cells or isolation of nuclei
By Wang Y-M et al. (2012) PLoS ONE 7(8): e42414.,

7. Comparison of technologies
Nature Reviews Genetics 13,   (December 2012)

9. SMiLE seq
Selective Microfluidics-based Ligand Enrichment followed by Sequencing
Quantitiative
Uses microfluidic device with TF transcribed/translated and modified with eGFP
DNA is labeled with Cy5
Use biotinylated eGFP Ab to bind to the TF(s)
Capture with streptavidin and remove unbound material
Capture DNA on membrane
Amplify and sequence
Nat Methods. 2017 Mar;14(3):
By Tessabw - Own work, CC BY-SA 4.0,

10. RNA binding protein-RNA interactions
HITS-CLIP (aka CLIP-seq)
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation
mapping protein–RNA binding sites or RNA modification sites in vivo
PAR-CLIP
photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation
identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs)

11. CLIP: RNA binding protein sites
By Bluewhale22 - Own work, CC BY-SA 3.0,

12. HITS-CLIP aka CLIP-seq
Adaptable to most systems; relatively easy to set up
UV light can cause mutations; low efficiency of cross-linking
By Bluewhale22 - Own work, CC BY-SA 3.0,

13. CLIP-seq to identify binding motif for TAF15

14. PAR-CLIP Increased resolution & decreased S/N over CLIP-seq
Can only be used in cell culture or single cell organisms
By Bluewhale22 - Own work, CC BY-SA 3.0,

15. In vitro motif identification with quantitation
RNA Bind-n-Seq
In vitro motif identification with quantitation
RBNS R is the motif read enrichment
RBFOX2 Canonical motif: GCAUG was determined by SELEX
Motif binding for Mbnl1

16. RNA Bind-n-Seq from Kapeli et. al.
GGUA in vitro motif with highest affinity for TAF15
GUGG in vitro motif with highest affinity for FUS

17. Combined RNA-seq and CLIP-seq
CLIP-seq identified targets of the RNA binding proteins (TAF15, FUS & TDP-43
RNA-seq identified transcripts with altered expression in absence of the three proteins
More overlap in the binding targets than in the differentially expressed genes
Genes downregulated by the ASO knockdowns of TAF15 or TDP-43 had more binding targets than did UP-regulated or unchanged genes

18. ENCODE (Encyclopedia of DNA elements)
Human, mouse, worm and fly genomes
Used computationally rather than via a browser
Trends in Genetics (2016) 32: 238–249

19. ENCODE data matrix

20. ENCODE tracks within UCSC genome browser