Advances in genomic technology lead to a more focused pattern for the distribution of chromosomal proteins and a better understanding of their functions. The recent development of the CUT&RUN technique marks one of the important such advances. Here we develop a modified CUT&RUN technique that we termed nanoCUT&RUN, in which a high affinity nanobody to GFP is used to bring micrococcal nuclease to the binding sites of GFP-tagged chromatin proteins. Subsequent activation of the nuclease cleaves the chromatin, and sequencing of released DNA identifies binding sites. We show that nanoCUT&RUN efficiently produces high quality data for the TRL transcription factor in Drosophila embryos, and distinguishes binding sites specific between two TRL isoforms. We further show that nanoCUT&RUN dissects the distributions of the HipHop and HOAP telomere capping proteins, and uncovers unexpected binding of telomeric proteins at centromeres. nanoCUT&RUN can be readily applied to any system in which a chromatin protein of interest, or its isoforms, carries the GFP tag. 

This repository contains all the coverage files produce in Chen, Wei, Courret, et al. 2022. They correspond to the distribution of HipHop, HOAP or WT (negative control) in the D. melanogaster genome, produced by nanoCUT&RUN. 

Briefly, we trimmed the reads with Trim Galore (https://github.com/FelixKrueger/TrimGalore/)  (paired end default settings), and then mapped the trimmed reads to a heterochromatin-enriched genome assembly (Chang and Larracuente 2019) using bowtie2  (Langmead et al. 2012). We defined uniquely mapped reads using samtools (v1.5) (-q 10). We then converted bam files to bigwig files (.bw) and normalized as RPM using the bamcoverage command from deeptools. 

References:

Chen, T., Wei, X., Courret, C., Cui, M., Cheng, L., Wu, J., Ahmad, K., Larracuente, A.M., Rong, Y. 2022. The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila. BIORXIV 4/22/22 https://www.biorxiv.org/content/10.1101/2022.04.20.488892v1. Forthcoming in PLOS Genetics.

Chang CH, Larracuente AM. Heterochromatin-Enriched Assemblies Reveal the Sequence and Organization of the Drosophila melanogaster Y Chromosome. Genetics. 2019 Jan;211(1):333-348.

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012 Mar 4;9(4):357-9. doi: 10.1038/nmeth.1923.

Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, Heyne S, Dündar F, Manke T. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Research. 2016 Apr 13:gkw257.

Bigwig files are indexed binary format. They can be open in IGV software to visualize the coverage track along the genome. We used them as input in our R script to make plots, using the package karyoploteR and the command kpPlotBigWig. 

Those coverage files were used to make Figure 2-4-S4-S5-S6-S10. The R scripts used to make the figures can be find here: https://github.com/LarracuenteLab/nanoCUTandRUN.github.

File are organized by figure. The first part of the file name corresponds to the genotype (HipHop or HOAP transgenes). The second part corresponds to the replicate number ("_1", "_2") or digestion time ("_2min", "_15min"). The last part of the file name indicates whether we kept multi-mapping reads (".mapped.sorted.RPM.bw) or only report uniquely mapping reads (".unique.mapped.sorted.RPM.bw"). For example "hiphop_2.mapped.sorted.RPM" corresponds to normalized multi-mapping read coverage of the second replicate of nanoCUT&Run data for the GFP-tagged HipHop strain. 

Files containing "_WG" correspond to the whole genome sequencing of the corresponding strain. For example "hiphop_WG.unique.mapped.sorted.RPM" correspond to the the normalized coverage of uniquely mapping reads of the whole genome sequencing of the GFP-tagged HipHop strain.