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

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

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

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

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., https://commons.wikimedia.org/w/index.php?curid=25629484

Comparison of technologies Nature Reviews Genetics 13, 840-852 (December 2012)

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):316-322. By Tessabw - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=56785789

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)

CLIP: RNA binding protein sites By Bluewhale22 - Own work, CC BY-SA 3.0, https://en.wikipedia.org/w/index.php?curid=42105218

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, https://commons.wikimedia.org/w/index.php?curid=31434082

CLIP-seq to identify binding motif for TAF15

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, https://commons.wikimedia.org/w/index.php?curid=31434083

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

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

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

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

ENCODE data matrix

ENCODE tracks within UCSC genome browser