The objective of this study was to investigate the mechanism that regulates pre-implantation development of the yak (Bos grunniens). We determined the transcriptomes of in vitro-produced yak embryos at two-cell, four-cell, eight-cell stages, and morula and blastocyst using the Illumina RNA-seq for the first time. We obtained 47.36-50.86 million clean reads for each stage, of which, 85.65%-90.02% reads were covered in the reference genome. A total of 17,368 genes were expressed during the two-cell stage to blastocyst of the yak, of which 7,236 genes were co-expressed at all stages, whereas 10,132 genes were stage-specific expression. Transcripts from 9,827 to 14,893 different genes were detected in various developmental stages. When |log ratio| ≥ 1 and q-value <0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 6,922-10,555 DEGs between any two consecutive stages. The GO distributions of these DEGs were classified into three categories: biological processes (23 terms), cellular components (22 terms) and molecular functions (22 terms). Pathway analysis revealed 310 pathways of the DEGs that were operative in early pre-implantation yak development, of which 32 were the significantly enriched pathways. In conclusion, this is the first report to investigate the mechanism that regulates yak embryonic development using high-throughput sequencing, which provides a comprehensive framework of transcriptome landscapes of yak pre-implantation embryos.

We describe a microfluidics-based strategy for genome-wide analysis of multiplex chromatin interactions with single-molecule precision. In multiplex chromatin interaction analysis (multi-ChIA), individual chromatin complexes are partitioned into droplets that contain a gel bead with unique DNA barcode, in which tethered chromatin DNA fragments are barcoded and amplified for sequencing and mapping to demarcate chromatin contacts. Thus, multi -ChIA has the unprecedented ability to uncover multiplex chromatin interactions at single -molecule level, which has been impossible using previous methods that rely on analyzing pairwise contacts via proximity ligation. We demonstrate that multiplex chromatin interactions predominantly contribute to topologically associated domains, and clusters of gene promoters and enhancers provide a fundamental topological framework for co-transcriptional regulation.Genomes of higher organisms from fly to human are known to be extensively folded into chromosomal territories within the three-dimensional (3D) nuclear space 1 . Advanced long-range chromatin interaction

11Summary: CRISPR-based methods for genome, epigenome editing and imaging have provided 12 powerful tools to interrogate the functions of the genome. The design of guide RNA (gRNA) is a 13 vital step of CRISPR experiments. We report here the implementation of JACKIE (Jackie and 14 Albert's CRISPR K-mer Instances Enumerator), a pipeline for enumerating all potential single-15 and multi-copy CRISPR sites in the genome. We demonstrate the application of JACKIE to 16 identify locus-specific repetitive sequences for CRISPR/Casilio-based genomic labeling. 17 18 Availability: Source codes and CRISPR site databases (JACKIEdb) for hg38 and mm10 are 19 available for download at http://crispr.software/JACKIE 20 21 118 ./, and score field recording the copy number.119 Parallelization is achieved by either starting separate job on individual <6merPrefix>.bin files, or 120 by starting batch jobs focusing on 2mer prefices (e.g., AA AT AC AG operating on AA*.bin, 121 AT*.bin, AC*.bin, AG*.bin, etc). The outputs (<6merPrefix>.bed) are then merged into a 122 combined bed file using cat unix command. Helper scripts are provided for downstream 123 processing of the bed file into collapsed bed file with each record encoding all binding sites of 124 6 the same sequence in the same chromosome, for extracting binding sites within defined 125 regions, or for running the Cas-OFFinder program to identify off-target profiles of selected 126 sequences. 127 Fig 2. Potential applications of JACKIE pipeline. (a) JACKIE can be used to identify 128 clustered CRISPR binding sites with high copy number or low copy number for CRISPR-based 129 genomic imaging experiments. (b) To demonstate genomic imaging use case, we filtered 130 sgRNA sequences with >30 copies clustered within specific <10kb regions and performed 131 Casilio imaging experiment using designed sgRNAs. UCSC genome browser screenshots are 132 shown on the left with the line-bar annotating the CRISPR sites, and fluorescence microscopy 133 images shown on the right. Microscopy images were derived by merging green (Casilio-134 labeling) and blue (DAPI: nucleus stain). Arrows point to fluorescent loci of interest. Scale bars 135 indicate 5μm. (c) JACKIE can be used to identify clustered CRISPR binding sites for synergistic 136 activation or repression of target cis-regulatory elements (e.g., promoters) using CRISPR-based 137 epigenetic editing approaches (e.g., CRISPRa or CRISPRi). (d) JACKIE can identify unique 138 CRISPR binding sites in the genome for precise indel induction, dual CRISPR sites for targeted 139 deletion, or clustered sites spanning a genomic region to induce complex rearrangement 140 events.

The three-dimensional genome structure plays a fundamental role in gene regulation 19 and cellular functions. Recent studies in genomics based on sequencing technologies inferred the 20 very basic functional chromatin folding structures of the genome known as chromatin loops, the 21 long-range chromatin interactions that are often mediated by protein factors. To visualize the 22 visualization of higher-order chromatin structures for the very short genomic segments can be 29 realized by microscopic imaging. 30 31 35 et al., 1997; Tsunaka et al., 2005) that are connected by dozens of bp of linker DNA, appearing as 36a "beads on a string" structure (Olins and Olins, 1974; Kornberg, 1974; Oudet et al., 1975; Finch 37 and Klug, 1976; Bustin et al., 1976; Leuba et al., 1994). The 10 nm "beads on a string" DNA fiber is 38 then folded into higher-order chromatin structures for further chromatin compaction. However, 39 1 of 13 Manuscript submitted to eLife the organization of higher-order chromatin structures was elusive for tens of years. Although the 40 30 nm chromatin fiber was observed and suggested to be the next organizational level of the 10 41 nm fiber, it is now debatable whether it exists in vivo (Felsenfeld and Groudine, 2003; van Holde 42 and Zlatanova, 2007; Nishino et al., 2012). Recently, technologies combining biochemistry and 43 high-throughput sequencing such as Hi-C (Lieberman-Aiden et al., 2009) and ChIA-PET (Fullwood 44 et al., 2009) have been developed to characterize genome-wide landscape of long-range chromatin 45 interactions (usually from several kilobases (kb) to hundreds of kilobases) that are considered as the 46 basis of higher-order chromatin organization. Chromatin interactions suggest the looping structure 47 119 the pre-processed images. We demonstrated a new image processing algorithm Figure 2-Figure 120 Supplement 2 that identifies the coordinates of the single molecules from pre-processed iPALM 121 images (Figure 1e) and returns points localizations in a PDB file format (Figure 2a), which can be 122 used in further modeling. Basically, we extracted significant signals from the images. We measured 123 the brightness of signals in relative luminosity units which range from 0 to 255. Brightness threshold 124 was chosen manually to cut off noise. In this way, we got different sets of dots for each image. 125 Borg I, Groenen PJ, Mair P. Applied multidimensional scaling and unfolding. Springer; 2017. 330 Bustin M, Goldblatt D, Sperling R. Chromatin structure visualization by immunoelectron microscopy. Cell. 1976; 331 7(2):297-304. 332 Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X. Evaluation of fluorophores for optimal performance in 333 localization-based super-resolution imaging. Nature methods. 2011; 8(12):domains in mammalian genomes 335 identified by analysis of chromatin interactions. Nature. 2012; 485(7398):376. 336 Felsenfeld G, Groudine M. Controlling the double helix. Nature. 2003; 421(6921):448. 337 Finch J, Klug A. Solenoidal model for superstructure in chromatin.